参见作者原研究论文

本实验方案简略版
May 2017

本文章节


 

Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
在上皮细胞中利用脱氧胆酸盐分离纤维粘连蛋白以及对回收的纤维粘连蛋白原纤维的生物素化分析    

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in MCF10A mammary epithelial and Caki-1 renal cancer epithelial cells. Using two detergent extractions, cellular FN is separated into detergent insoluble or fibril incorporated FN and soluble FN or unincorporated fractions. To determine whether fibrillogenesis utilizes a recycled pool of FN, we have used a Biotin labeled FN (FN-Biotin) recycling assay, that has been modified from a previous study. Using a combination of the recycling assay and deoxycholate fractionation methods, one can quantitatively demonstrate the extent of fibrillogenesis in cells under different experimental conditions and determine the source of FN for fibrillogenesis.

Keywords: Fibronectin (FN) (纤维粘连蛋白), Fibrillogenesis (原纤维形成), Extracellular matrix (细胞外基质), Recycling (回收), Endocytosis (内吞作用)

Background

Fibronectin (FN) is a ubiquitously produced extra cellular matrix (ECM) component (Uitto et al., 1989; Mao and Schwarzbauer, 2005). Fibronectin pools are generated transcriptionally that can be increased by several growth factors such as TGF-β1 (Yokoi et al., 2002; Mimura et al., 2004; Tang et al., 2007). The step-wise process of fibrillogenesis involving cell surface receptor Integrin α5β1 engagement with dimeric FN, drives the process of fibrillogenesis in cells (Yang and Hynes, 1996). Integrin α5β1 regulation by receptor activation/inactivation cycles, receptor endocytosis and recycling influences fibrillogenesis (Gao et al., 2000; White et al., 2007; Caswell et al., 2008) with FN contributing to net endocytosis rates of integrins in active conformations (Arjonen et al., 2012). The relevance of FN fibrillogenesis to cellular outcome has been demonstrated by numerous studies investigating fibril-specific functions for the FN protein. For example, a polymeric form of fibronectin or ‘super-fibronectin’ shows anti-metastatic and anti-angiogenic properties against different tumor types (Pasqualini et al., 1996; Yi and Ruoslahti, 2001). In the absence of a FN matrix, as observed in Von Hippel Lindau syndrome, renal cancer characterized by the mutation or loss of the Von Hippel Lindau (VHL) protein (Ohh et al., 1998; Hoffman et al., 2001), introducing a VHL mutant unable to form a fibronectin matrix is insufficient to suppress formation of tumors in SCID mice (Stickle et al., 2004). All disease mutants of the VHL gene in renal cancer also fail to form a FN matrix (Hoffman et al., 2001). Thus, analyses of fibril versus soluble FN maybe of importance to investigations that explore the contribution of the FN matrix to cellular response. Endpoint and real-time assays have been successfully used to study fibrillogenesis (Pankov et al., 2000; Mao and Schwarzbauer, 2005). Both studies rely on microscopy-based approaches to determine fibrillogenesis in cells. Biochemical fractional of FN is a quantitative approach to complement microscopy-based methods to detect levels of fibril incorporated from soluble FN. Using a fractionation assay in combination with a recycling assay, allows us to determine whether FN that is incorporated in the matrix is recycled from an existing pool of FN in the cell or from cell autonomous sources. The FN-Biotin recycling assay is simpler than classical temperature-switching assays that investigate receptor recycling (Roberts et al., 2001). Additionally, the temperature-switching assays quantify protein recycling from a decrease in rate of protein endocytosis; requiring additional lysosomal or proteasomal inhibitors in the assay. Our recycling assay can be adapted to many different proteins that localize intracellularly and at the cell membrane. While performing our FN fractionation protocol we did detect integrin β1 in the soluble and insoluble fractions. It is possible to extend this observation to perform recycling of integrins using a Biotin-conjugated integrin or any protein of interest. Alternatively, it is also possible to perform immunoprecipitation experiments on the recycled protein fraction to determine whether specific proteins interact preferentially with the two FN fractions. This protocol can help answer several questions associated with protein trafficking kinetics, interacting partner proteins and new functional characteristics based on associating proteins in the different fractions.

Materials and Reagents

  1. 15 ml conical tube (Thermo Fisher Scientific, catalog number: 339650 )
  2. Pipette tip
    1,250 μl (USA Scientific, TipOne, catalog number: 1112-1720 )
    200 μl (USA Scientific, TipOne, catalog number: 1110-1700 )
    20 μl (USA Scientific, TipOne, catalog number: 1123-1710 )
    10 μl (USA Scientific, TipOne, catalog number: 1111-3700 )
  3. Aluminum foil (Walmart, 551605957)
  4. Beaker (100 ml, WWR, catalog number: 890000-200 )
  5. Filter paper (GE Healthcare, catalog number: 1001-929 )
  6. T75 flask (Corning, catalog number: 353824 )
  7. Six-well tissue culture plates (Corning, catalog number: 3506 )
  8. Cell lifter (Fisher Scientific, FisherbrandTM, catalog number: 08-100-240 )
  9. 23 G needle (BD, Precision Glide, catalog number: 305193 )
  10. Microcentrifuge tubes (Eppendorf, catalog number: 022600028 )
  11. MCF10A breast cell lines (ATCC, catalog number: CRL-10317 )
  12. Caki-1 renal cancer cell lines (ATCC, catalog number: HTB-46 )
  13. Protein Ladder (Bio-Rad Laboratories, catalog number: 1610375 )
  14. PVDF protein transfer membrane (Merck, catalog number: IPVH08100 )
  15. Fibronectin antibody (Santa Cruz Biotechnology, catalog number: sc-59826 )
  16. Actin antibody (Abcam, catalog number: ab8227 )
  17. GAPDH antibody (Abcam, catalog number: ab9484 )
  18. Biotin labeled fibronectin (CYTOSKELETON, catalog number: FNR03 )
  19. Streptavidin conjugated IRDye® (LI-COR, catalog number: 926-32230 )
  20. IRDye 800CW Secondary Antibody (LI-COR, catalog number: 925-32210 )
  21. DMEM/F-12 HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 11330057 )
  22. Insulin (Thermo Fisher Scientific, GibcoTM, catalog number: 12585014 )
  23. EGF (Thermo Fisher Scientific, GibcoTM, catalog number: PHG0313 )
  24. Hydrocortisone (Corning, catalog number: 354203 )
  25. Horse Serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16050122 )
  26. Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  27. Cholera toxin (Sigma-Aldrich, catalog number: C8052-2MG )
  28. Skimmed Milk powder (Thermo Fisher Scientific, catalog number: LP0031B )
  29. BSA (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP9703100 )
  30. McCoy's 5A (ATCC, catalog number: 30-2007 )
  31. FBS (Corning, catalog number: 35-011-CV )
  32. 0.05% Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300062 )
  33. SDS (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP166-100 )
  34. Sodium deoxycholate (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP349-100 )
  35. Tris Base (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP152-1 )
  36. Tris-HCl (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP153-1 )
  37. Glycine (Fisher Scientific, Fisher ChemicalTM, catalog number: G48-212 )
  38. Tween 20 (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP337-500 )
  39. Sodium Hydroxide pellets (Fisher Scientific, Fisher ChemicalTM, catalog number: S318-1 )
  40. Ethanol, absolute (200 proof) (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP2818500 )
  41. N-ethylmaleimide (Thermo Fisher Scientific, catalog number: 23030 )
  42. Iodoacetic acid (Thermo Fisher Scientific, catalog number: 35603 )
  43. DTT (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP172-25 )
  44. Bromophenol blue (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP115-25 )
  45. Nitrocellulose (GE Healthcare, catalog number: 10600012 )
  46. NaCl (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP358-1 )
  47. KCl (Fisher Scientific, Fisher ChemicalTM, catalog number: P217-3 )
  48. Na2HPO4 (MP Biomedicals, catalog number: 02191440.1 )
  49. KH2PO4 (MP Biomedicals, catalog number: 02195453.1 )
  50. 100% Methanol (Fisher Scientific, Fisher ChemicalTM, catalog number: A935-4 )
  51. Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
  52. Phenylmethylsulphonyl fluoride (PMSF) (ACROS ORGANICS, catalog number: 215740050 )
  53. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: 5312-500 )
  54. Growth media for MCF10A cells (see Recipes)
  55. Growth media for Caki-1 cells (see Recipes)
  56. 10x PBS (see Recipes)
  57. 1x PBS (see Recipes)
  58. 10x TBS (see Recipes)
  59. 1x TBS (see Recipes)
  60. 1x TBS 0.2% Tween (see Recipes)
  61. 1x SDS Running Buffer (see Recipes)
  62. 1x Transfer Buffer (see Recipes)
  63. 2 M DTT (see Recipes)
  64. 5x Loading Dye (see Recipes)
  65. Blocking Buffer (see Recipes)
  66. Primary antibody buffer (see Recipes)
  67. Secondary antibody buffer (see Recipes)
  68. Acid wash (see Recipes)
  69. 1 M Tris-HCl pH 8.0 (see Recipes)
  70. 1 M Tris-HCl pH 8.8 (see Recipes)
  71. 100 mM Phenylmethylsulphonyl fluoride (PMSF) (see Recipes)
  72. 100 mM Iodoacetic acid (see Recipes)
  73. 100 mM N-Ethylmaleimide (NEM) (see Recipes)
  74. 0.5 M Ethylenediaminetetraacetic acid (EDTA) pH 8.0 (see Recipes)
  75. Deoxycholate lysis buffer (see Recipes)
  76. SDS lysis buffer (see Recipes)

Equipment

  1. Magnetic stirrer (Corning, catalog number: 440936 )
  2. Rotor wheel (Thermo Fisher Scientific, catalog number: 88881001 )
  3. Heating block (Thermo Fisher Scientific, catalog number: 88870001 )
  4. Orbital Shaker (Corning, catalog number: 6780-FP )
  5. Microcentrifuge (Eppendorf, model: 5424 R , catalog number: 5404000138)
  6. Centrifuge (Thermo Fisher Scientific, model: SorvallTM Legend T plus , catalog number: 75004367)
  7. Incubator (PHC, Panasonic, model: MCO-170AICUVL-PA )
  8. Pipettes (5 ml; 10 ml, Fisher Scientific, FisherBrandTM, catalog numbers: 13-676-10C ; 13-676-10F )
  9. Tissue culture hood (Thermo Fisher Scientific, catalog number: 1333 )
  10. Autoclave
  11. Odyssey Fc Imaging System (LI-COR, model number: 2800 )
  12. Light microscope (Zeiss, model: ID03 )
  13. Ice bucket
  14. -20 °C freezer (Fisher Scientific, catalog number: 13-986-148 )
  15. SDS PAGE apparatus and Transfer tank (Bio-Rad Laboratories, catalog number: 1658029fc )
  16. Tray to set up transfer (12 ½" x 17 ½")

Software

  1. LI-COR Lite (https://www.licor.com/bio/products/software/image_studio_lite/download.html)
  2. ImageJ (https://imagej.nih.gov/ij/download.html)
  3. Microsoft excel

Procedure

  1. Deoxycholate fractionation of FN in MCF10A and Caki-1 cells
    1. MCF10A breast cell lines or Caki-1 renal cancer cells should be maintained to reach no more than 80-90% confluence in a T75 tissue culture flask in a 5% CO2 buffered incubator. Refer Figure 1 for the quick start protocol.
    2. Once the cells are approximately 80% confluent in the flask, aspirate the cell culture medium, rinse the cells once in 15 ml 1x PBS and aspirate before adding 2-3 ml 0.05% Trypsin-EDTA. Place the flask in a 5% CO2 buffered incubator for 5-7 min.
    3. Once the cells are successfully dislodged from the flask which you can check using a light microscope, add 3 ml of growth medium to the flask to neutralize the action of trypsin.
    4. Transfer the 6 ml cell suspension into a 15 ml conical tube and centrifuge at 300 x g for 5 min at room temperature.
    5. After centrifugation, discard the supernatant and resuspend the pellet in growth medium.
    6. For plating in 6-well dishes, plate cells at a confluence that will reach 80% on the day of the experiment. For Caki-1 cells the plating number is 100,000 cells/well and MCF10A cells 70,000 cells/well. Cells in one well of the six-well plate are sufficient to visualize the detergent soluble and insoluble FN fractions for each experimental condition.
      Note: Cell density of 80% provides sufficient soluble and insoluble FN protein for quantification. If cell density is a variable in your experimental condition, the 80% density is ideal since cells are not too confluent to trigger cellular pathways that may interfere with your experimental read-out.
    7. Incubate the cells at 37 °C at 5% CO2.
    8. The following day, check to see whether optimum cell density has been reached. If yes, the cells can be processed for deoxycholate fractionation of FN.
      Note: Since the entire fractionation procedure is performed on ice, it is important at this stage that you have cold 1x PBS, cold deoxycholate lysis buffer (inhibitors freshly added) and cold SDS lysis buffer (inhibitors freshly added). Refer ‘Recipes’ section.
    9. Aspirate medium from wells and place the plates on ice in an ice bucket. Add 10 ml 1x cold PBS to remove any trace of growth medium in the cells and aspirate completely. Rinse again with 1x PBS if you see traces of media in the well. It is important at this point to make sure all PBS is aspirated so that subsequent addition of lysis buffer is not diluted.
    10. To each well, add 300 μl of cold (4 °C) deoxycholate lysis buffer containing inhibitors and immediately scrape the cells using a cell lifter.
    11. Transfer all the cells in the lysis buffer into an Eppendorf tube and lyse using a 23 G syringe needle at least 20 times.
    12. Place the tubes on a rotor wheel in the cold room and allow to rotate for 30 min. Rotor speed is not critical for lysis. Lysis time can be extended to 1 h at this point without affecting results (optional).
    13. Transfer the Eppendorf tubes to a microfuge and centrifuge at 21,130 x g for 30 min at 4 °C. If the microfuge has a maximum speed of 15,871 x g, you can increase the centrifugation time to 45 min.
    14. Check to see if you spot a pellet in the size of a pinhead. The pellets are usually very difficult to see and sometimes require a second round of centrifugation.
      Note: It is also a good idea to place the Eppendorf tubes in the microfuge and mark the cap of the tube where the pellet will settle. This makes it easier to know where to look for the pellet.
    15. Transfer the supernatant to a new Eppendorf tube and label it ‘soluble FN’. This protein fraction contains detergent soluble FN. To the soluble FN fraction, add 5x SDS loading dye and store in a -20 °C freezer until ready to resolve proteins on a gel.
    16. To the pellet, add 20 μl cold SDS lysis buffer containing inhibitors and mix the pellet with the buffer using a pipette tip.
    17. Heat the pellet to 95 °C for 1 min for the pellet to mix completely with the SDS lysis buffer. This protein fraction contains the deoxycholate ‘insoluble FN’.
      Note: Heating the pellet for 1 min with SDS lysis buffer is a necessary step to completely resuspend the pellet.
    18. Add 5x SDS loading dye, mix and store in the -20 °C freezer. 
    19. Before loading ‘soluble FN and ‘insoluble FN’ samples on an SDS-PAGE gel, heat samples to 95 °C for 5 min and centrifuge at 21,130 x g at RT for 1 min.


      Figure 1. Flowchart for extraction and separation of the two FN fractions

  2. Biotinylation assay to measure recycled FN in MCF10A cells
    1. The MCF10A cells are seeded under the same conditions and incubated in a 5% CO2 buffered incubator as described in Procedure A.
    2. Reconstitute one vial of lyophilized FN-Biotin with 10 μl sterile distilled water and allow the tube to sit for a few minutes at room temperature before tapping the tube a few times to mix. The concentration of the stock will now be 2 mg/ml.
    3. Add 20 μl of the stock solution to 2 ml serum free media to make a working concentration of 20 μg/ml FN-Biotin medium. To make 2 ml serum free media containing a working concentration of 20 μg/ml FN-Biotin, you will need a total of two vials of lyophilized FN-Biotin. In our hands, reducing the working concentration to 10 μg/ml proved insufficient to determine FN uptake by MCF10A cells using immunoblotting.
    4. Before beginning the biotinylation assay, serum starve the cells for 2 h. Serum starvation is performed by aspirating growth media from the cells, washing cells two times each with 2 ml 1x PBS and adding serum-free medium to the cells.
    5. After the 2 h incubation, aspirate the serum-free medium from cells, wash cells in 6-well plates twice each with 2 ml 1x PBS, aspirate PBS completely and add 500 μl of FN-Biotin media to the cells.
    6. Incubate for 30 min in the 37 °C incubator.
    7. After incubation, aspirate the FN-Biotin medium, wash with 2 ml 1x PBS, aspirate and add 1 ml acid wash (see Recipes) for 30 sec. The acid wash step is critical to remove cell surface bound FN-Biotin. It is important not to exceed acid wash to more than 1 min as it begins to affect cell morphology.
    8. Remove the acid wash and rinse the well four times each with 2 ml 1x PBS.
    9. Aspirate the PBS and add growth medium to the cells without FN-Biotin and with or without test compounds depending on your experimental question. Since we were interested to investigate whether TGFβ1 and TGFβ2 increases FN fibrillogenesis via recycling, we had a control condition with only growth medium, growth medium containing 10 ng/ml TGFβ1 and growth medium containing 10 ng/ml TGFβ2 (Varadaraj et al., 2017).
    10. To determine whether FN is recycled to form fibrils, perform a deoxycholate fractionation as described in Procedure A, Steps 8-17, using 300 μl of deoxycholate lysis buffer per well.

  3. Resolving the FN protein on a 5% gel and immunoblotting
    1. Run the soluble and pellet lysates on a 5% SDS-PAGE gel.
    2. Load 1/10th of the total volume of soluble fraction and the entire volume of the pellet fraction. If you load 1/10th, keep the volume consistent for all experimental conditions.
    3. Let the gel run till only the top two ladders of the protein ladder are remaining within the gel -250 kDa and 150 kDa. The gel run usually takes 3-4 h at 80 V.
    4. After the run is complete, transfer the gel using either Nitrocellulose or PVDF membranes at 25 V overnight (12-16 h) in a cold room. Alternatively, the transfer tank can be placed in an ice bucket during the transfer procedure.
    5. After the transfer is complete, place the membrane in an immunoblotting box and add 5 ml of blocking buffer (sufficient volume to cover the membrane) for 1 h at room temperature and place on an orbital shaker at 55 rpm.
    6. After blocking, remove the blocking solution and wash the membrane 2-3 times with 1x TBS using gentle agitation. Make sure all traces of milk has left the membrane. Rinse until the TBS washes are clear.
    7. Add FN antibody at a dilution of 1:1,000 in primary antibody buffer and allow the membrane to incubate overnight on an orbital shaker set at a speed of 55 rpm at 4 °C.
    8. After primary antibody incubation, rinse three times by placing the membrane on an orbital shaker set at a speed of 55 rpm for 5 min in 10 ml 1x TBS 0.2% Tween.
    9. Add IR Dye 800CW secondary antibody at a dilution of 1 μl in 15 ml secondary antibody buffer for 1 h on an orbital shaker set at a speed of 55 rpm. It is important to protect the membrane from light. If the immunoblot boxes are transparent, cover the box with aluminum foil throughout the incubation and subsequent wash steps.
    10. To visualize FN-Biotin, perform Steps C1-C6 following which add Streptavidin conjugated IR Dye secondary antibody at a dilution of 1 μl in 15 ml secondary antibody buffer for 1 h on an orbital shaker set at a speed of 55 rpm. It is important to protect the membrane from light. If the immunoblot boxes are transparent, cover the box with aluminum foil throughout the incubation and subsequent wash steps.
    11. After antibody incubation, rinse three times by placing the membrane on an orbital shaker set at a speed of 55 rpm for 5 min in 10 ml 1x TBS 0.2% Tween.
    12. Remove 1x TBS 0.2% Tween and replace with 10 ml 1x TBS before imaging on the LI-COR scanner.
    13.  An image scan of the membrane will appear as shown in Figure 2A.
    14. To probe for a loading control protein, run 1/10th of the soluble FN fractions on a 10% gel and immunoblot for GAPDH (Figure 2B) or Actin. Actin and GAPDH primary antibodies are used at a dilution of 1 μl in 5 ml (1 μg/5 ml) and can be reused several times if the diluted antibody is stored at 4 °C.
      Note: HRP conjugated secondary antibodies can also be used and processed using an ECL detection method. In that case, you will need an HRP conjugated Streptavidin secondary antibody.


      Figure 2. Fibronectin fractionation and analysis by immunoblotting. To visualize and quantify soluble and pellet FN, load 1/10th of the soluble and the entire pellet fractions from Caki-1 cells, on a 5% SDS-PAGE gel as shown in (A). A band above the 250 kDa MW ladder is FN. UN, 1, 2 and 3 refer to ‘untreated’ and three different treatment conditions (B). To normalize for protein loading, load 1/10th of the soluble FN on a 10% SDS-PAGE gel and immunoblot for GAPDH. Pixel intensities of the protein bands are quantified using the LI-COR Lite software by drawing a rectangle (blue box) around the band. The lower value denotes the background-corrected band intensity and the upper value denotes the background.

Data analysis

Typically, deoxycholate extractions and biotinylation assays are tested independently at least three times to get statistically significant results. To quantify FN that is in fibrils (pellet fraction) compared to the non-fibril (soluble FN) fraction, immunoblots of resolved FN and GAPDH are required. Follow the below step-by-step bullet points to perform data analysis of the samples.

  1. Using the LI-COR Lite software, quantify all the protein bands individually and export to an excel sheet.
  2. If the experimental question is to test whether FN fibril increases with treatment, calculate the pixel intensities of soluble FN to corresponding GAPDH or Actin levels.
  3. After normalizing soluble FN levels, calculate the ratio of pellet FN pixel intensities to the normalized soluble FN levels. In Figure 2A, soluble FN in UN (untreated) and treatment conditions 1, 2 and 3 are normalized to GAPDH in UN, 1, 2 and 3 respectively.
  4. Calculate pixel intensities for pellet FN in UN, 1, 2 and 3.
  5. Finally calculate ratios for UN pellet FN versus normalized UN soluble FN, sample 1 pellet FN versus normalized sample 1 soluble FN and so on.
  6. To represent the analyzed data as a figure in a publication, convert the ratios to fold difference/relative difference, create a bar graph of the fold difference between UN and treated samples, the UN sample being ‘1’, calculate SEM and perform appropriate statistics (Refer Figure 4E in Varadaraj et al., 2017). 

If the data clearly demonstrates that pellet FN increases with treatment X, it is likely that both soluble and pellet FN increase, in which case you may wrongly infer that only the pellet FN is increasing. To check if this were the case, it is essential to perform the same experiment but extract total protein instead of deoxycholate fractions. If the total FN levels remain the same between treatments, then you can conclude that only the fibril fractions are changing with treatments. Alternatively, if the total FN protein increases with treatment, then the increase in pellet FN may be due to an increase in the total protein and not specifically a difference in pellet FN fraction between treatments. Our study (refer Figure 1C in Varadaraj et al., 2017) showed that TGFβ1 and TGFβ2 increases FN fibril fraction, we measured total FN levels in parallel experiments to fractionation experiments to confirm that FN fibril fraction was increasing with treatments.

Recipes

  1. Growth media for MCF10A cells
    1. To 500 ml of DMEM/F12 media:
      Add 25 ml of horse serum, 1.25 ml of Insulin from a 4 mg/ml Insulin stock solution, 350 μl of Cholera toxin from 150 μg/ml stock, 215 μl of EGF from 50 μg/ml stock and 500 μl Hydrocortisone from 500 μg/ml stock
    2. To make stock solution of Cholera toxin:
      Dissolve 2 mg Cholera toxin in 13.3 ml DMEM/F12 (without additives) and store as 350 μl aliquots in the -20 °C freezer. We have used aliquots of Cholera toxin that has been stored for no longer than 6 months
    3. To make EGF stock solution:
      Dissolve 1 mg lyophilized EGF in 20 ml sterile dH2O and store as 215 μl aliquots in the -20 °C freezer
    4. To make Hydrocortisone stock solution:
      Dissolve 50 mg Hydrocortisone in 100 ml 200 proof ethanol and store as 10 ml aliquots in the -20 °C freezer
  2. Growth media for Caki-1 cells
    To 500 ml McCoy's 5A medium add 50 ml FBS and 5 ml Penicillin-Streptomycin (optional)

Note: *These buffers can be stored at room temperature.

  1. 10x PBS (1 L) *
    Weigh 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4 and add 500 ml sterile dH2O and adjust pH using HCl to pH 7.2 to 7.4
    Make up the final volume to 1 L using sterile dH2O
  2. 1x PBS (1 L) *
    Dilute 100 ml of 10x PBS with 900 ml sterile dH2O
    10x TBS (1L) *
    1. Weigh 87.66 g NaCl, 12.11 g Tris base and add 500 ml sterile dH2O
    2. Adjust pH using HCl to pH 8.0 and make up final volume to 1 L using sterile dH2O
  3. 1x TBS (1 L) *
    Dilute 1 part of 10x TBS with 9 parts sterile dH2O
  4. 1x TBS 0.2% Tween (1 L) *
    Add 2 ml Tween 20 to 1x TBS
  5. 1x SDS Running Buffer (10 L) *
    1. Add 303 g Tris Base, 1,440 g Glycine and 100 g SDS
    2. Make up to a total volume of 10 L using sterile dH2O
  6. 1x Transfer Buffer (1 L)
    Add 1.86 g Glycine, 3.02 g Tris Base, 150 ml 100% Methanol and bring upto a total volume of 1 L with dH2O
    Note: The transfer buffer can be stored at 4 °C.
  7. 2 M DTT
    Weigh 3.08 g of DTT (FW 154.25) in 10 ml sterile dH2O
    Aliquot and store in the -20 °C freezer
  8. 5x Loading Dye
    1. To make a total volume of 100 ml loading dye:
      1. Add 50 ml glycerol to a beaker, 10 g SDS, 33 ml 1 M Tris pH 6.8 and 17 ml water
      2. Allow the solution to mix by placing on a magnetic stirrer set at 37 °C for the SDS to dissolve completely
      3. Pick a small amount of Bromophenol blue powder using a pipette tip and mix into the solution
    2. To 90 ml of this solution add 10 ml of 2 M DTT
    3. Store as 2 ml aliquots in the -20 °C freezer
  9. Blocking Buffer
    Weigh 0.25 g skimmed milk powder and dissolve in 1x TBS
    Prepare 10 ml fresh for each use
  10. Primary antibody buffer
    Weigh 0.05 g BSA and dissolve in 1x TBS
    Prepare 10 ml fresh for each use
  11. Secondary antibody buffer
    Weigh 0.05 g BSA and dissolve in 1x TBS 0.2% Tween
    Prepare 10 ml fresh for each use
  12. Acid wash
    Weigh 14.6 g NaCl and add 2.5 ml glacial acetic acid
    Dilute with sterile dH2O up to 500 ml and store at room temperature
  13. 1 M Tris-HCl pH 8.0 (1 L) *
    1. Weigh 157.6 g of Tris-HCl salt and dissolve in 900 ml sterile dH2O
    2. After the salt has dissolved, adjust pH using NaOH to pH 8.0 and add water up to a total of 1 L
  14. 1 M Tris-HCl pH 8.8 (1 L) *
    1. Weigh 157.6 g of Tris-HCl salt and dissolve in 900 ml sterile dH2O
    2. After the salt has dissolved, adjust pH using NaOH to pH 8.8 and add water up to a total of 1 L
  15. 100 mM Phenylmethylsulphonyl fluoride (PMSF)
    Weigh 0.174 g in 10 ml ethanol to obtain a 100 mM stock solution (FW 174.2)
    Aliquot and store in the -20 °C freezer
  16. 100 mM Iodoacetic acid
    Weigh 0.185 g in 10 ml sterile dH2O to obtain a 100 mM stock solution (FW 185.95)
    Aliquot and store in the -20 °C freezer
  17. 100 mM N-Ethylmaleimide (NEM)
    Weigh 0.125 g in 10 ml ethanol to obtain a 100 mM stock solution (FW 125.13)
    Aliquot and store in the -20 °C freezer
  18. 0.5 M Ethylenediaminetetraacetic acid (EDTA) pH 8.0 (FW 292.24) (0.5 L) *
    1. Weigh 73.06 g and add 400 ml sterile dH2O
    2. Allow the salt to dissolve and adjust pH to 8.0
    3. Add water to make up to a final volume of 500 ml
  19. Deoxycholate lysis buffer (0.1 L)
    2% Sodium Deoxycholate
    0.02 M Tris-HCl pH 8.8
    2 mM PMSF
    2 mM EDTA
    2 mM Iodoacetic Acid
    2 mM N-ethylmalemide
    1. To make 100 ml buffer:
      Add 2 g Sodium deoxycholate salt, 2 ml 1 M Tris-HCl pH 8.8 and remaining sterile dH2O upto a total volume of 100 ml
      Note: This buffer can be stored in the refrigerator for no more than 3 months.
    2. To make 10 ml of this buffer with inhibitors:
      Add 20 μl of 100 mM PMSF stock, 40 μl of 0.5 M EDTA stock, 20 μl Iodoacetic acid from 100 mM stock and 20 μl NEM from 100 mM stock
      Note: Inhibitors should be added freshly before use at final concentrations of 2 mM PMSF, 2 mM EDTA pH 8.0, 2 mM Iodoacetic Acid and 2 mM NEM.
  20. SDS lysis buffer (0.1 L) *
    1. To make 100 ml buffer:
      Add 1 g SDS and 2.5 ml 1 M Tris-HCl pH 8.0 and water up to 100 ml
    2. For 10 ml of the SDS lysis buffer:
      Add 20 μl of 100 mM PMSF stock, 40 μl of 0.5 M EDTA stock, 20 μl Iodoacetic acid from 100 mM stock and 20 μl NEM from 100 mM stock
      Note: Inhibitors should be added freshly before use at final concentrations of 2 mM PMSF, 2 mM EDTA pH 8.0, 2 mM Iodoacetic Acid and 2 mM NEM.

Acknowledgments

Research reported in this publication was funded in part by National Institute On Minority Health and Health Disparities of the National Institutes of Health under Award Number U54MD012388 and National Cancer Institute of the National Institutes of Health under the awards for the Partnership of Native American Cancer Prevention U54CA143924 (UACC) and U54CA143925 (NAU) to AV and National Institutes of Health grant P20 GM109091 to K.M. We would also like to acknowledge the work from Jean Schwarzbauer (Princeton University) and Donaldson, J.G (National Institutes of Health) that helped develop protocols for our studies.

Competing interests

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Authors declare no conflicts of interest or competing interests.

References

  1. Arjonen, A., Alanko, J., Veltel, S. and Ivaska, J. (2012). Distinct recycling of active and inactive β1 integrins. Traffic 13(4): 610-625.
  2. Caswell, P. T., Chan, M., Lindsay, A. J., McCaffrey, M. W., Boettiger, D. and Norman, J. C. (2008). Rab-coupling protein coordinates recycling of α5β1 integrin and EGFR1 to promote cell migration in 3D microenvironments. J Cell Biol 183(1): 143-155.
  3. Gao, B., Curtis, T. M., Blumenstock, F. A., Minnear, F. L. and Saba, T. M. (2000). Increased recycling of α5β1 integrins by lung endothelial cells in response to tumor necrosis factor. J Cell Sci 113 Pt 2: 247-257.
  4. Hoffman, M. A., Ohh, M., Yang, H., Klco, J. M., Ivan, M. and Kaelin, W. G., Jr. (2001). von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF. Hum Mol Genet 10(10): 1019-1027.
  5. Mao, Y. and Schwarzbauer, J. E. (2005). Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol 24(6): 389-399.
  6. Mimura, Y., Ihn, H., Jinnin, M., Asano, Y., Yamane, K. and Tamaki, K. (2004). Epidermal growth factor induces fibronectin expression in human dermal fibroblasts via protein kinase C delta signaling pathway. J Invest Dermatol 122(6): 1390-1398.
  7. Ohh, M., Yauch, R. L., Lonergan, K. M., Whaley, J. M., Stemmer-Rachamimov, A. O., Louis, D. N., Gavin, B. J., Kley, N., Kaelin, W. G., Jr. and Iliopoulos, O. (1998). The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol Cell 1(7): 959-968. 
  8. Pankov, R., Cukierman, E., Katz, B. Z., Matsumoto, K., Lin, D. C., Lin, S., Hahn, C. and Yamada, K. M. (2000). Integrin dynamics and matrix assembly: tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis. J Cell Biol 148(5): 1075-1090.
  9. Pasqualini, R., Bourdoulous, S., Koivunen, E., Woods, V. L., Jr. and Ruoslahti, E. (1996). A polymeric form of fibronectin has antimetastatic effects against multiple tumor types. Nat Med 2(11): 1197-1203.
  10. Roberts, M., Barry, S., Woods, A., van der Sluijs, P. and Norman, J. (2001). PDGF-regulated rab4-dependent recycling of αvβ3 integrin from early endosomes is necessary for cell adhesion and spreading. Curr Biol 11(18): 1392-1402. 
  11. Stickle, N. H., Chung, J., Klco, J. M., Hill, R. P., Kaelin, W. G., Jr. and Ohh, M. (2004). pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development. Mol Cell Biol 24(8): 3251-3261.
  12. Tang, C. H., Yang, R. S., Chen, Y. F. and Fu, W. M. (2007). Basic fibroblast growth factor stimulates fibronectin expression through phospholipase C γ, protein kinase C α, c-Src, NF-κB, and p300 pathway in osteoblasts. J Cell Physiol 211(1): 45-55.
  13. Uitto, J., Olsen, D. R. and Fazio, M. J. (1989). Extracellular matrix of the skin: 50 years of progress. J Invest Dermatol 92(4 Suppl): 61S-77S.
  14. Varadaraj, A., Jenkins, L. M., Singh, P., Chanda, A., Snider, J., Lee, N. Y., Amsalem-Zafran, A. R., Ehrlich, M., Henis, Y. I. and Mythreye, K. (2017). TGF-β triggers rapid fibrillogenesis via a novel TbetaRII-dependent fibronectin-trafficking mechanism. Mol Biol Cell 28(9): 1195-1207.
  15. White, D. P., Caswell, P. T. and Norman, J. C. (2007). αvβ3 and α5β1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration. J Cell Biol 177(3): 515-525.
  16. Yang, J. T. and Hynes, R. O. (1996). Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins. Mol Biol Cell 7(11): 1737-1748.
  17. Yi, M. and Ruoslahti, E. (2001). A fibronectin fragment inhibits tumor growth, angiogenesis, and metastasis. Proc Natl Acad Sci U S A 98(2): 620-624.
  18. Yokoi, H., Mukoyama, M., Sugawara, A., Mori, K., Nagae, T., Makino, H., Suganami, T., Yahata, K., Fujinaga, Y., Tanaka, I. and Nakao, K. (2002). Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol 282(5): F933-942.

简介

纤连蛋白(FN)是一种细胞外基质蛋白,由许多细胞类型分泌,主要与细胞表面受体整合素α5β1结合。整合素α5β1结合启动FN逐步组装成原纤维,这一过程称为原纤维形成。我们和其他几个人已经证明了原纤维形成对细胞迁移和转移的关键作用。虽然免疫染色和显微镜方法有助于可视化FN掺入原纤维,每个原纤维的长度至少为3μm,但是第一项研究开发了一种生物化学分离FN以量化原纤维并入FN的方法,由Jean Schwarzbauer小组于1996年出版。我们的方案改编自原始出版物,并已在多种细胞类型上进行测试,包括如此处所示的MCF10A乳腺上皮细胞和Caki-1肾癌上皮细胞。使用两种洗涤剂提取物,将细胞FN分离成不溶于洗涤剂或掺入原纤维的FN和可溶性FN或未掺入的级分。为了确定原纤维形成是否利用FN的再循环池,我们使用了生物素标记的FN(FN-生物素)再循环测定,其已经从先前的研究中修改。使用再循环测定和脱氧胆酸盐分离方法的组合,可以定量地证明在不同实验条件下细胞中原纤维形成的程度,并确定原纤维形成的FN来源

【背景】 纤连蛋白(FN)是普遍产生的细胞外基质(ECM)组分(Uitto et al。,1989; Mao和Schwarzbauer,2005)。纤连蛋白库是转录产生的,可以通过几种生长因子如TGF-β1增加(Yokoi et al。,2002; Mimura et al。,2004; Tang 等人,2007)。涉及细胞表面受体整合素α5β1与二聚体FN结合的原纤维形成的逐步过程驱动细胞中原纤维形成的过程(Yang和Hynes,1996)。通过受体激活/失活循环,受体内吞作用和再循环调节整合素α5β1影响原纤维形成(Gao et al。,2000; White et al。,2007; Caswell et al。,2008)FN有助于活性构象中整合素的净胞吞作用率(Arjonen et al。,2012)。 FN原纤维形成与细胞结果的相关性已经通过研究FN蛋白的原纤维特异性功能的大量研究得到证实。例如,聚合形式的纤连蛋白或'超纤连蛋白'显示出针对不同肿瘤类型的抗转移和抗血管生成特性(Pasqualini 等人,1996; Yi和Ruoslahti,2001)。在没有FN基质的情况下,如在Von Hippel Lindau综合征中所观察到的,肾癌的特征在于Von Hippel Lindau(VHL)蛋白的突变或丧失(Ohh et al。,1998; Hoffman et al。,2001),引入 VHL 突变体不能形成纤维连接蛋白基质不足以抑制SCID小鼠肿瘤的形成(Stickle 等。。,2004)。肾癌中 VHL 基因的所有疾病突变体也不能形成FN基质(Hoffman 等人,,2001)。因此,原纤维与可溶性FN的分析对于探索FN基质对细胞反应的贡献的研究可能是重要的。终点和实时检测已成功用于研究原纤维形成(Pankov et al。,2000; Mao和Schwarzbauer,2005)。两项研究都依赖于基于显微镜的方法来确定细胞中的原纤维形成。 FN的生物化学分数是用于补充基于显微镜的方法的定量方法,以检测从可溶性FN掺入的原纤维的水平。使用分馏分析与循环分析相结合,我们可以确定掺入基质中的FN是从细胞中现有的FN池还是从细胞自主来源回收的。 FN-生物素再循环测定比调查受体再循环的经典温度转换测定更简单(Roberts et al。,2001)。此外,温度转换试验通过降低蛋白质内吞作用的速率来量化蛋白质回收。在测定中需要额外的溶酶体或蛋白酶体抑制剂。我们的回收试验可以适应许多不同的蛋白质,这些蛋白质在细胞内和细胞膜上定位。在执行我们的FN分级方案时,我们确实检测了可溶性和不溶性级分中的整联蛋白β1。可以延长该观察结果以使用生物素缀合的整联蛋白或任何目的蛋白质进行整联蛋白的再循环。或者,还可以对再循环的蛋白质级分进行免疫沉淀实验,以确定特定蛋白质是否优先与两种FN级分相互作用。该协议可以帮助回答与蛋白质运输动力学相关的几个问题,相互作用的伴侣蛋白质和基于不同组分中的蛋白质相关的新功能特征。

关键字:纤维粘连蛋白, 原纤维形成, 细胞外基质, 回收, 内吞作用

材料和试剂

  1. 15毫升锥形管(Thermo Fisher Scientific,目录号:339650)
  2. 移液器吸头
    1,250μl(USA Scientific,TipOne,目录号:1112-1720)
    200μl(USA Scientific,TipOne,目录号:1110-1700)
    20μl(USA Scientific,TipOne,目录号:1123-1710)
    10μl(USA Scientific,TipOne,目录号:1111-3700)
  3. 铝箔(Walmart,551605957)
  4. 烧杯(100毫升,WWR,目录号:890000-200)
  5. 滤纸(GE Healthcare,目录号:1001-929)
  6. T75烧瓶(康宁,目录号:353824)
  7. 六孔组织培养板(Corning,目录号:3506)
  8. 细胞提升器(Fisher Scientific,Fisherbrand TM ,目录号:08-100-240)
  9. 23 G针(BD,Precision Glide,目录号:305193)
  10. 微量离心管(Eppendorf,目录号:022600028)
  11. MCF10A乳腺细胞系(ATCC,目录号:CRL-10317)
  12. Caki-1肾癌细胞系(ATCC,目录号:HTB-46)
  13. Protein Ladder(Bio-Rad Laboratories,目录号:1610375)
  14. PVDF蛋白转移膜(默克,目录号:IPVH08100)
  15. Fibronectin抗体(Santa Cruz Biotechnology,目录号:sc-59826)
  16. 肌动蛋白抗体(艾博抗(上海)贸易有限公司,目录号:ab8227)
  17. GAPDH抗体(Abcam,目录编号:ab9484)
  18. 生物素标记的纤连蛋白(CYTOSKELETON,目录号:FNR03)
  19. 链霉抗生物素蛋白缀合的IRDye ®(LI-COR,目录号:926-32230)
  20. IRDye 800CW二抗(LI-COR,目录号:925-32210)
  21. DMEM / F-12 HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:11330057)
  22. 胰岛素(Thermo Fisher Scientific,Gibco TM ,目录号:12585014)
  23. EGF(Thermo Fisher Scientific,Gibco TM ,目录号:PHG0313)
  24. 氢化可的松(Corning,目录号:354203)
  25. 马血清(Thermo Fisher Scientific,Gibco TM ,目录号:16050122)
  26. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  27. 霍乱毒素(Sigma-Aldrich,目录号:C8052-2MG)
  28. 脱脂奶粉(赛默飞世尔科技,目录号:LP0031B)
  29. BSA(Fisher Scientific,Fisher BioReagents TM ,目录号:BP9703100)
  30. McCoy的5A(ATCC,目录号:30-2007)
  31. FBS(Corning,目录号:35-011-CV)
  32. 0.05%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25300062)
  33. SDS(Fisher Scientific,Fisher BioReagents TM ,目录号:BP166-100)
  34. 脱氧胆酸钠(Fisher Scientific,Fisher BioReagents TM ,目录号:BP349-100)
  35. Tris Base(Fisher Scientific,Fisher BioReagents TM ,目录号:BP152-1)
  36. Tris-HCl(Fisher Scientific,Fisher BioReagents TM ,目录号:BP153-1)
  37. 甘氨酸(Fisher Scientific,Fisher Chemical TM ,目录号:G48-212)
  38. 吐温20(Fisher Scientific,Fisher BioReagents TM ,目录号:BP337-500)
  39. 氢氧化钠颗粒(Fisher Scientific,Fisher Chemical TM ,目录号:S318-1)
  40. 绝对乙醇(200标准)(Fisher Scientific,Fisher BioReagents TM ,目录号:BP2818500)
  41. N-乙基马来酰亚胺(赛默飞世尔科技,目录号:23030)
  42. 碘乙酸(赛默飞世尔科技,目录号:35603)
  43. DTT(Fisher Scientific,Fisher BioReagents TM ,目录号:BP172-25)
  44. Bromophenol blue(Fisher Scientific,Fisher BioReagents TM ,目录号:BP115-25)
  45. 硝化纤维素(GE Healthcare,目录号:10600012)
  46. NaCl(Fisher Scientific,Fisher BioReagents TM ,目录号:BP358-1)
  47. KCl(Fisher Scientific,Fisher Chemical TM ,目录号:P217-3)
  48. Na 2 HPO 4 (MP Biomedicals,目录号:02191440.1)
  49. KH 2 PO 4 (MP Biomedicals,目录号:02195453.1)
  50. 100%甲醇(Fisher Scientific,Fisher Chemical TM ,目录号:A935-4)
  51. 冰醋酸(Fisher Scientific,目录号:A38-212)
  52. 苯基甲基磺酰氟(PMSF)(ACROS ORGANICS,目录号:215740050)
  53. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:5312-500)
  54. MCF10A细胞的生长培养基(见食谱)
  55. Caki-1细胞的生长培养基(见食谱)
  56. 10x PBS(见食谱)
  57. 1x PBS(见食谱)
  58. 10倍TBS(见食谱)
  59. 1x TBS(见食谱)
  60. 1x TBS 0.2%吐温(见食谱)
  61. 1x SDS运行缓冲液(参见食谱)
  62. 1x转移缓冲液(见食谱)
  63. 2 M DTT(见食谱)
  64. 5倍装载染料(见食谱)
  65. 阻塞缓冲液(见食谱)
  66. 一抗缓冲液(见食谱)
  67. 二抗缓冲液(见食谱)
  68. 酸洗(见食谱)
  69. 1 M Tris-HCl pH 8.0(参见配方)
  70. 1 M Tris-HCl pH 8.8(见食谱)
  71. 100 mM苯基甲基磺酰氟(PMSF)(见食谱)
  72. 100毫摩尔碘乙酸(见食谱)
  73. 100 mM N-乙基马来酰亚胺(NEM)(参见食谱)
  74. 0.5 M乙二胺四乙酸(EDTA)pH 8.0(见食谱)
  75. 脱氧胆酸盐裂解缓冲液(见食谱)
  76. SDS裂解缓冲液(见食谱)

设备

  1. 磁力搅拌器(康宁,目录号:440936)
  2. 转子轮(Thermo Fisher Scientific,目录号:88881001)
  3. 加热块(Thermo Fisher Scientific,目录号:88870001)
  4. Orbital Shaker(康宁,目录号:6780-FP)
  5. Microcentrifuge(Eppendorf,型号:5424 R,目录号:5404000138)
  6. 离心机(Thermo Fisher Scientific,型号:Sorvall TM Legend T plus,目录号:75004367)
  7. 孵化器(PHC,Panasonic,型号:MCO-170AICUVL-PA)
  8. 移液管(5ml; 10ml,Fisher Scientific,FisherBrand TM ,目录号:13-676-10C; 13-676-10F)
  9. 组织培养罩(Thermo Fisher Scientific,目录号:1333)
  10. 高压灭菌器
  11. Odyssey Fc成像系统(LI-COR,型号:2800)
  12. 光学显微镜(蔡司,型号:ID03)
  13. 冰桶
  14. -20°C冰柜(Fisher Scientific,目录号:13-986-148)
  15. SDS PAGE装置和转移罐(Bio-Rad Laboratories,目录号:1658029fc)
  16. 托盘设置转移(12½“x17½”)

软件

  1. LI-COR Lite( https://www.licor.com/bio/产品/软件/ image_studio_lite / download.html )
  2. ImageJ( https://imagej.nih.gov/ij/download.html )
  3. 微软擅长

程序

  1. MCF10A和Caki-1细胞中FN的脱氧胆酸盐分馏
    1. 应保持MCF10A乳腺细胞系或Caki-1肾癌细胞在5%CO 2 缓冲培养箱中的T75组织培养瓶中达到不超过80-90%的汇合。有关快速启动协议,请参阅图1。
    2. 一旦细胞在烧瓶中约80%汇合,吸出细胞培养基,用15ml 1x PBS冲洗细胞一次并吸出,然后加入2-3ml 0.05%胰蛋白酶-EDTA。将烧瓶置于5%CO 2 缓冲培养箱中5-7分钟。
    3. 一旦细胞从烧瓶中成功移出,您可以使用光学显微镜检查,向烧瓶中加入3 ml生长培养基以中和胰蛋白酶的作用。
    4. 将6ml细胞悬浮液转移到15ml锥形管中,并在室温下以300×g离心5分钟离心。
    5. 离心后,弃去上清液并将沉淀重悬于生长培养基中。
    6. 对于6孔培养皿中的平板培养,在实验当天将达到80%的汇合处的平板细胞。对于Caki-1细胞,电镀数为100,000个细胞/孔,MCF10A细胞为70,000个细胞/孔。对于每种实验条件,六孔板的一个孔中的细胞足以可视化洗涤剂可溶性和不溶性FN级分。
      注意:80%的细胞密度提供足够的可溶性和不溶性FN蛋白用于定量。如果细胞密度在实验条件下是一个变量,那么80%的密度是理想的,因为细胞不会太融合而不能触发可能干扰实验读数的细胞通路。
    7. 将细胞在37℃,5%CO 2 下孵育。
    8. 第二天,检查是否达到最佳细胞密度。如果是,可以处理细胞用于FN的脱氧胆酸盐分馏。
      注意:由于整个分馏过程是在冰上进行的,因此在这个阶段重要的是你有冷的1x PBS,冷脱氧胆酸盐裂解缓冲液(新鲜添加的抑制剂)和冷SDS裂解缓冲液(新添加的抑制剂)。请参阅“食谱”部分。
    9. 从孔中吸出培养基并将板置于冰桶中的冰上。加入10ml 1x冷PBS以除去细胞中任何痕量的生长培养基并完全吸出。如果在孔中看到痕量的培养基,则用1x PBS再次冲洗。此时重要的是确保吸出所有PBS,以便随后加入裂解缓冲液不被稀释。
    10. 向每个孔中加入300μl含有抑制剂的冷(4℃)脱氧胆酸盐裂解缓冲液,并立即用细胞提升器刮擦细胞。
    11. 将裂解缓冲液中的所有细胞转移到Eppendorf管中,并使用23G注射器针头溶解至少20次。
    12. 将管放在冷室中的转子轮上,并旋转30分钟。转子速度对于裂解并不重要。此时裂解时间可延长至1小时而不影响结果(可选)。
    13. 将Eppendorf管转移至微量离心机并在21,130 x g 下在4℃下离心30分钟。如果微量离心机的最大速度为15,871 x g ,则可以将离心时间增加到45分钟。
    14. 检查是否发现了针头大小的颗粒。颗粒通常很难看到,有时需要第二轮离心。
      注意:将Eppendorf管放入微量离心机中并标记颗粒将沉淀在其中的管帽也是一个好主意。这样可以更容易地知道在哪里寻找颗粒。
    15. 将上清液转移到新的Eppendorf管中并将其标记为“可溶性FN”。该蛋白质级分含有可溶于洗涤剂的FN。向可溶性FN级分中加入5x SDS上样染料并储存在-20℃冰箱中直至准备好在凝胶上分离蛋白质。
    16. 向沉淀中加入含有抑制剂的20μl冷SDS裂解缓冲液,并使用移液管尖端将沉淀物与缓冲液混合。
    17. 将沉淀加热至95℃1分钟,使沉淀与SDS裂解缓冲液完全混合。该蛋白质部分含有脱氧胆酸盐'不溶性FN'。
      注意:用SDS裂解缓冲液加热沉淀1分钟是完全重悬颗粒的必要步骤。
    18. 加入5x SDS上样染料,混合并储存在-20°C冰箱中。 
    19. 在将可溶性FN和'不溶性FN'样品加载到SDS-PAGE凝胶上之前,将样品加热至95℃5分钟,并在室温下以21,130 x g 离心1分钟。


      图1.提取和分离两种FN级分的流程图

  2. 生物素化测定法测量MCF10A细胞中回收的FN
    1. 如步骤A所述,在相同条件下接种MCF10A细胞,并在5%CO 2 缓冲培养箱中孵育。
    2. 用10μl无菌蒸馏水重新配制一小瓶冻干的FN-生物素,并使管在室温下静置几分钟,然后将管轻敲几次以混合。现在浓度为2毫克/毫升。
    3. 将20μl储备溶液加入2ml无血清培养基中,使工作浓度为20μg/ ml FN-生物素培养基。要制备含有工作浓度为20μg/ ml FN-生物素的2 ml无血清培养基,您需要总共两瓶冻干的FN-生物素。在我们的手中,将工作浓度降低至10μg/ ml证明不足以使用免疫印迹确定MCF10A细胞对FN的摄取。
    4. 在开始生物素化测定之前,血清使细胞饥饿2小时。通过从细胞中吸出生长培养基,用2ml 1x PBS洗涤细胞两次并向细胞中加入无血清培养基来进行血清饥饿。
    5. 孵育2小时后,从细胞中吸出无血清培养基,用2ml 1x PBS洗涤6孔板中的细胞,每次2ml,完全吸出PBS,并向细胞中加入500μlFN-生物素培养基。
    6. 在37°C培养箱中孵育30分钟。
    7. 孵育后,吸出FN-生物素培养基,用2ml 1x PBS洗涤,吸出并加入1ml酸洗液(见食谱)30秒。酸洗步骤对于去除细胞表面结合的FN-生物素是至关重要的。重要的是不要超过酸洗超过1分钟,因为它开始影响细胞形态。
    8. 取出酸洗液,用2 ml 1x PBS冲洗孔四次。
    9. 根据您的实验问题,吸出PBS并在没有FN-生物素和有或没有测试化合物的情况下向细胞中添加生长培养基。由于我们有兴趣研究TGFβ1和TGFβ2是否通过循环增加FN原纤维形成,我们只有生长培养基,含有10 ng /mlTGFβ1的生长培养基和含有10 ng /mlTGFβ2的生长培养基的对照条件(Varadaraj et al 。,2017)。
    10. 为了确定FN是否被再循环形成原纤维,按照程序A,步骤8-17中所述进行脱氧胆酸盐分级分离,每孔使用300μl脱氧胆酸盐裂解缓冲液。

  3. 在5%凝胶上解析FN蛋白并进行免疫印迹
    1. 在5%SDS-PAGE凝胶上运行可溶性和沉淀裂解物。
    2. 加载可溶部分总体积的1/10 th 和颗粒部分的总体积。如果加载1/10 th ,请保持所有实验条件的体积一致。
    3. 让凝胶运行直到只有蛋白质梯子的顶部两个梯子保留在凝胶-250kDa和150kDa内。凝胶运行通常需要3-4小时,电压为80 V.
    4. 运行完成后,使用硝酸纤维素膜或PVDF膜在25V下在冷室中转移凝胶过夜(12-16h)。或者,转移罐可以在转移过程中放置在冰桶中。
    5. 转移完成后,将膜置于免疫印迹盒中,并在室温下加入5ml封闭缓冲液(足以覆盖膜的体积)1小时,并置于55rpm的轨道振荡器上。
    6. 封闭后,取出封闭溶液,用1x TBS洗涤膜2-3次,轻轻搅拌。确保所有牛奶痕迹都留下了膜。冲洗直至TBS洗涤清洁。
    7. 在第一抗体缓冲液中以1:1,000稀释度添加FN抗体,并使膜在轨道振荡器上以55rpm的速度在4℃下孵育过夜。
    8. 在一抗孵育后,通过将膜置于10转1x TBS 0.2%Tween中以55rpm的速度设置的定轨振荡器上冲洗3次。
    9. 在设定为55rpm的定轨振荡器上,以1μl稀释度在15ml二抗缓冲液中加入IR Dye 800CW二抗1小时。保护膜免受光照很重要。如果免疫印迹盒是透明的,则在整个孵育和随后的洗涤步骤中用铝箔覆盖盒子。
    10. 为了使FN-生物素可视化,进行步骤C1-C6,然后在15ml二级抗体缓冲液中以1μl的稀释度在轨道振荡器上以55rpm的速度添加链霉抗生物素蛋白缀合的IR染料二抗1小时。保护膜免受光照很重要。如果免疫印迹盒是透明的,则在整个孵育和随后的洗涤步骤中用铝箔覆盖盒子。
    11. 在抗体孵育后,通过将膜置于10转1x TBS 0.2%Tween中以55rpm的速度设定的轨道振荡器上冲洗3次。
    12. 在LI-COR扫描仪上成像之前,取出1x TBS 0.2%吐温并更换为10 ml 1x TBS。
    13.  膜的图像扫描将如图2A所示。
    14. 为了探测上样对照蛋白,在10%凝胶上运行1/10 th 的可溶性FN级分,并对GAPDH(图2B)或肌动蛋白进行免疫印迹。肌动蛋白和GAPDH一抗在5 ml(1μg/ 5 ml)中以1μl的稀释度使用,如果稀释的抗体在4°C下储存,可以重复使用几次。
      注意:HRP偶联的二抗也可以使用ECL检测方法进行处理和处理。在这种情况下,您需要HRP共轭链霉抗生物素蛋白二抗。


      图2.纤连蛋白分级分离和免疫印迹分析。为了可视化和定量可溶性和沉淀FN,在5%SDS-PAGE凝胶上加载来自Caki-1细胞的可溶性和全部沉淀级分的1/10 ,如(A)中所示。高于250 kDa MW梯的频段是FN。 UN,1,2和3指的是“未经处理”和三种不同的处理条件(B)。为了标准化蛋白质加载,在10%SDS-PAGE凝胶上加载1/10 th 的可溶性FN,并对GAPDH进行免疫印迹。使用LI-COR Lite软件通过在带周围绘制矩形(蓝色框)来量化蛋白质条带的像素强度。较低的值表示背景校正的带强度,较高的值表示背景。

数据分析

通常,脱氧胆酸盐提取和生物素化测定至少独立地测试三次以获得统计学上显着的结果。为了量化原纤维中的FN(沉淀部分)与非原纤维(可溶性FN)部分相比,需要解析的FN和GAPDH的免疫印迹。按照以下逐步的要点进行样品的数据分析。

  1. 使用LI-COR Lite软件,单独量化所有蛋白质条带并导出至excel表。
  2. 如果实验问题是测试FN原纤维是否随治疗而增加,则计算可溶性FN的像素强度至相应的GAPDH或肌动蛋白水平。
  3. 在归一化可溶性FN水平后,计算颗粒FN像素强度与标准化可溶性FN水平的比率。在图2A中,UN中的可溶性FN(未处理)和处理条件1,2和3分别归一化为UN中的GAPDH,1,2和3。
  4. 计算UN,1,2和3中颗粒FN的像素强度。
  5. 最后计算UN颗粒FN与标准化UN可溶性FN,样品1颗粒FN与标准化样品1可溶性FN等的比率。
  6. 要将分析数据表示为出版物中的图形,请将比率转换为折叠差异/相对差异,创建UN和处理样本之间折叠差异的条形图,UN样本为“1”,计算SEM并执行适当的统计(参见Varadaraj et al。,2017年的图4E)。 

如果数据清楚地表明颗粒FN随着处理X的增加而增加,那么可溶性和颗粒FN都可能增加,在这种情况下,您可能错误地推断出只有颗粒FN正在增加。为了检查是否是这种情况,必须进行相同的实验,但提取总蛋白质而不是脱氧胆酸盐部分。如果治疗之间的总FN水平保持不变,那么您可以得出结论,只有原纤维部分随治疗而变化。或者,如果总FN蛋白质随处理而增加,则颗粒FN的增加可能是由于总蛋白质的增加,而不是处理之间颗粒FN部分的差异。我们的研究(参见Varadaraj et al。,2017中的图1C)显示TGFβ1和TGFβ2增加FN原纤维分数,我们在平行实验中测量总FN水平进行分级实验以确认FN原纤维分数增加治疗。

食谱

  1. MCF10A细胞的生长培养基
    1. 至500毫升DMEM / F12培养基:
      从4 mg / ml胰岛素原液中加入25 ml马血清,1.25 ml胰岛素,来自150μg/ ml原液的350μl霍乱毒素,来自50μg/ ml原液的215μlEGF和来自500μg的500μl氢化可的松/ ml库存
    2. 制作霍乱毒素的储备液:
      将2mg霍乱毒素溶于13.3ml DMEM / F12(不含添加剂)中,并在-20℃冰箱中以350μl等分试样储存。我们使用了等分的霍乱毒素,储存时间不超过6个月
    3. 制作EGF原液:
      将1 mg冻干的EGF溶于20 ml无菌dH 2 O中,并在-20°C冰箱中以215μl等分试样储存
    4. 制作氢化可的松原液:
      将50毫克氢化可的松溶于100毫升200标准乙醇中,并在-20°C冰箱中以10毫升等分试样储存
  2. Caki-1细胞的生长培养基
    向500毫升McCoy的5A培养基中加入50毫升FBS和5毫升青霉素 - 链霉素(可选)

    *注意:这些缓冲区可以在室温下存储。

  3. 10x PBS(1升)*
    称取80g NaCl,2g KCl,14.4g Na 2 HPO 4 ,2.4g KH 2 PO 4 加入500ml无菌dH 2 O并用HCl调节pH至pH7.2至7.4
    使用无菌dH 2 O将最终体积补足至1L
  4. 1x PBS(1升)*
    用900ml无菌dH 2 O稀释100ml 10x PBS 10x TBS(1L)*
    1. 称取87.66g NaCl,12.11g Tris碱,加入500ml无菌dH 2 O
    2. 使用HCl将pH调节至pH 8.0,并使用无菌dH 2 O将最终体积补足至1L
  5. 1x TBS(1升)*
    用9份无菌dH 2 O稀释1份10x TBS
  6. 1x TBS 0.2%吐温(1升)*
    将2ml Tween 20加入1x TBS中
  7. 1x SDS运行缓冲液(10 L)*
    1. 加入303g Tris碱,1,440g甘氨酸和100g SDS
    2. 使用无菌dH 2 O补足总体积10L
  8. 1x转移缓冲液(1 L)
    加入1.86克甘氨酸,3.02克Tris碱,150毫升100%甲醇,加入总体积1升,dH 2 O
    注意:传输缓冲区可以存储在4°C。
  9. 2 M DTT
    在10毫升无菌dH 2 O中称取3.08克DTT(FW 154.25)。 分装并储存在-20°C冰箱中
  10. 5倍装载染料
    1. 使总体积为100毫升的染料:
      1. 向烧杯,10g SDS,33ml 1M Tris pH 6.8和17ml水中加入50ml甘油
      2. 通过置于设定在37℃的磁力搅拌器使溶液混合,使SDS完全溶解
      3. 使用移液管尖端挑取少量溴酚蓝粉末并混合到溶液中
    2. 向90ml该溶液中加入10ml 2M DTT
    3. 在-20°C冰箱中以2 ml等分试样储存
  11. 阻塞缓冲区
    称取0.25克脱脂奶粉,溶于1x TBS中 每次使用准备10毫升新鲜
  12. 一抗缓冲液
    称取0.05 g BSA并溶解于1x TBS中 每次使用准备10毫升新鲜
  13. 二抗缓冲液
    称取0.05g BSA并溶于1x TBS 0.2%Tween中 每次使用准备10毫升新鲜
  14. 酸洗
    称取14.6 g NaCl并加入2.5 ml冰醋酸
    用无菌dH 2 O稀释至500 ml并在室温下储存
  15. 1 M Tris-HCl pH 8.0(1 L)*
    1. 称取157.6g Tris-HCl盐并溶于900ml无菌dH 2 O中
    2. 盐溶解后,使用NaOH调节pH至pH 8.0并加水至总共1升
  16. 1 M Tris-HCl pH 8.8(1 L)*
    1. 称取157.6g Tris-HCl盐并溶于900ml无菌dH 2 O中
    2. 盐溶解后,使用NaOH调节pH至pH8.8并加水至总共1L
  17. 100mM苯基甲基磺酰氟(PMSF)
    在10ml乙醇中称取0.174g,得到100mM储备溶液(FW 174.2) 分装并储存在-20°C冰箱中
  18. 100 mM碘乙酸
    在10ml无菌dH 2 O中称取0.185g,得到100mM原液(FW 185.95)
    分装并储存在-20°C冰箱中
  19. 100mM N-乙基马来酰亚胺(NEM)
    在10ml乙醇中称取0.125g,得到100mM储备溶液(FW 125.13) 分装并储存在-20°C冰箱中
  20. 0.5 M乙二胺四乙酸(EDTA)pH 8.0(FW 292.24)(0.5 L)*
    1. 称重73.06g并加入400ml无菌dH 2 O.
    2. 让盐溶解并将pH调节至8.0
    3. 加水使最终体积达到500毫升
  21. 脱氧胆酸盐裂解缓冲液(0.1 L)
    2%脱氧胆酸钠
    0.02 M Tris-HCl pH 8.8
    2 mM PMSF
    2 mM EDTA
    2 mM碘乙酸
    2mM N-乙基马来酰胺
    1. 制作100毫升缓冲液:
      加入2克脱氧胆酸钠盐,2毫升1M Tris-HCl pH 8.8和剩余的无菌dH 2 O至总体积100毫升
      注意:此缓冲液可以存放在冰箱中不超过3个月。
    2. 用抑制剂制备10毫升这种缓冲液:
      加入20μl100mM PMSF原液,40μl0.5M EDTA原液,20μlIldoaceticacid(100 mM原液)和20μlNEM(100 mM原液)
      注意:使用前应先添加抑制剂,最终浓度为2 mM PMSF,2 mM EDTA pH 8.0,2 mM碘乙酸和2 mM NEM。
  22. SDS裂解缓冲液(0.1 L)*
    1. 制作100毫升缓冲液:
      加入1 g SDS和2.5 ml 1M Tris-HCl pH 8.0,加水至100 ml
    2. 对于10ml SDS裂解缓冲液:
      加入20μl100mM PMSF原液,40μl0.5M EDTA原液,20μlIldoaceticacid(100 mM原液)和20μlNEM(100 mM原液)
      注意:使用前应先添加抑制剂,最终浓度为2 mM PMSF,2 mM EDTA pH 8.0,2 mM碘乙酸和2 mM NEM。

致谢

本出版物中报告的研究部分由美国国立卫生研究院少数民族健康与卫生差异国家研究所授予,奖项编号为U54MD012388,美国国立卫生研究院国家癌症研究所颁发美国原住民癌症预防合作奖U54CA143924 (UACC)和U54CA143925(NAU)向AV和国立卫生研究院授予P20 GM109091至KM内容完全由作者负责,并不一定代表国立卫生研究院的官方观点。我们还要感谢Jean Schwarzbauer(普林斯顿大学)和Donaldson,J.G(国立卫生研究院)帮助制定研究方案的工作。
作者声明没有利益冲突或竞争利益。

参考

  1. Arjonen,A.,Alanko,J.,Veltel,S。和Ivaska,J。(2012)。 有效和无效β1整合素的独特回收。 流量 13(4):610-625。
  2. Caswell,P.T.,Chan,M.,Lindsay,A.J。,McCaffrey,M.W。,Boettiger,D。和Norman,J.C。(2008)。 Rab偶联蛋白协调α5β1整合素和EGFR1的循环,促进3D微环境中的细胞迁移。 J Cell Biol 183(1):143-155。
  3. Gao,B.,Curtis,T.M.,Blumenstock,F.A.,Minnear,F.L。和Saba,T.M。(2000)。 增加肺内皮细胞对α5β1整合素的循环,以应对肿瘤坏死因子。 J Cell Sci 113 Pt 2:247-257。
  4. Hoffman,M.A.,Ohh,M.,Yang,H.,Klco,J.M.,Ivan,M。和Kaelin,W。G.,Jr。(2001)。 与2C型VHL疾病相关的von Hippel-Lindau蛋白突变体保留了下调HIF的能力。 Hum Mol Genet 10(10):1019-1027。
  5. Mao,Y。和Schwarzbauer,J。E.(2005)。 纤连蛋白原纤维形成,细胞介导的基质组装过程。 Matrix Biol < / em> 24(6):389-399。
  6. Mimura,Y.,Ihn,H.,Jinnin,M.,Asano,Y.,Yamane,K。和Tamaki,K。(2004)。 表皮生长因子通过蛋白激酶C delta信号通路诱导人皮肤成纤维细胞中纤维连接蛋白的表达。 J Invest Dermatol 122(6):1390-1398。
  7. Ohh,M.,Yauch,RL,Lonergan,KM,Whaley,JM,Stemmer-Rachamimov,AO,Louis,DN,Gavin,BJ,Kley,N.,Kaelin,WG,Jr。和Iliopoulos,O。(1998) 。 von Hippel-Lindau肿瘤抑制蛋白是正确组装细胞外纤连蛋白基质所必需的。 Mol Cell 1(7):959-968。&nbsp;
  8. Pankov,R.,Cukierman,E.,Katz,B.Z.,Matsumoto,K.,Lin,D.C.,Lin,S.,Hahn,C.and Yamada,K.M。(2000)。 整合素动力学和基质组装:α5β1整合素的张力依赖性易位促进早期纤维连接蛋白原纤维形成。 J Cell Biol 148(5):1075-1090。
  9. Pasqualini,R.,Bourdoulous,S.,Koivunen,E.,Woods,VL,Jr。和Ruoslahti,E。(1996)。聚合形式的纤连蛋白对多种肿瘤类型具有抗转移作用。 Nat Med 2(11):1197-1203。
  10. Roberts,M.,Barry,S.,Woods,A.,van der Sluijs,P。和Norman,J。(2001)。 PDGF调节的rab4依赖性回收早期内体中的αvβ3整合素是细胞粘附和扩散所必需的。 Curr Biol 11(18):1392-1402。&nbsp;
  11. Stickle,N.H.,Chung,J.,Klco,J.M.,Hill,R.P.,Kaelin,W.G.,Jr。和Ohh,M。(2004)。 NEDD8的pVHL修饰是纤连蛋白基质组装和抑制肿瘤发展所必需的。 Mol Cell Biol 24(8):3251-3261。
  12. Tang,C.H.,Yang,R。S.,Chen,Y.F。和Fu,W。M.(2007)。 碱性成纤维细胞生长因子通过磷脂酶Cγ,蛋白激酶Cα,c-Src刺激纤连蛋白的表达, NF-κB和成骨细胞中的p300途径。 J Cell Physiol 211(1):45-55。
  13. Uitto,J.,Olsen,D。R.和Fazio,M。J.(1989)。 皮肤细胞外基质:50年的进展。 J Invest Dermatol 92(4 Suppl):61S-77S。
  14. Varadaraj,A.,Jenkins,LM,Singh,P.,Chanda,A.,Snider,J.,Lee,NY,Amsalem-Zafran,AR,Ehrlich,M.,Henis,YI和Mythreye,K。(2017) 。 TGF-β通过新的TbetaRII依赖性纤连蛋白 - 运输机制触发快速原纤维形成。 Mol Biol Cell 28(9):1195-1207。
  15. White,D。P.,Caswell,P。T.和Norman,J。C.(2007)。 αvβ3和α5β1整合素循环途径决定下游Rho激酶信号传导,以调节持续性细胞迁移。 J Cell Biol 177(3):515-525。
  16. Yang,J。T.和Hynes,R。O.(1996)。 缺乏α5β1整合素的胚胎细胞中的纤连蛋白受体功能可被αV整联蛋白取代。 Mol Biol Cell 7(11):1737-1748。
  17. Yi,M。和Ruoslahti,E。(2001)。 纤连蛋白片段抑制肿瘤生长,血管生成和转移。 Proc Natl Acad Sci USA 98(2):620-624。
  18. Yokoi,H.,Mukoyama,M.,Sugawara,A.,Mori,K.,Nagae,T.,Makino,H.,Suganami,T.,Yahata,K.,Fujinaga,Y.,Tanaka,I。and Nakao,K。(2002)。 结缔组织生长因子在纤连蛋白表达和肾小管间质纤维化中的作用。 Am J Physiol Renal Physiol 282(5):F933-942。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Varadaraj, A., Magdaleno, C. and Mythreye, K. (2018). Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells. Bio-protocol 8(16): e2972. DOI: 10.21769/BioProtoc.2972.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。