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May 2020

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Intracellular IRF5 Dimerization Assay
细胞内IRF5二聚分析   

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Abstract

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5 dimers, THP-1 cells are incubated in the presence or absence of the IRF5-targeted CPP (IRF5-CPP) inhibitor for 30 min then the cells are stimulated with R848 for 1 h. Cell lysates are separated by native-polyacrylamide gel electrophoresis (PAGE) and IRF5 dimers are detected by immunoblotting with IRF5 antibodies. To detect endogenous interactions between IRF5 and FITC-labeled IRF5-CPP, an in-cell fluorescence resonance energy transfer (FRET) assay is used. In this assay, THP-1 cells are left untreated or treated with FITC-IRF5-CPP conjugated inhibitors for 1 h. Next, cells are fixed, permeabilized, and stained with anti-IRF5 and TRITC-conjugated secondary antibodies. Transfer of fluorescence can be measured and calculated as FRET units. These methods provide rapid and accurate assays to detect IRF5 molecular interactions.

Keywords: IRF5 (细胞内干扰素调节因子5), Native-PAGE (非变性聚丙烯酰胺凝胶电泳), Polyacrylamide gel electrophoresis (聚丙烯酰胺凝胶电泳), FRET (荧光共振能量转移), Dimerization (二聚作用), Molecular interaction (分子间相互作用), IRF5-CPP (干扰素调节因子5-细胞穿透肽)

Background

Interferon regulatory factor 5 (IRF5) is a transcription factor that regulates pathogen-induced innate and acquired immune responses downstream of Toll-like receptor (TLR), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and B cell receptor (BCR) (De et al., 2017; Thompson et al., 2018; Banga et al., 2020). IRF5 has been implicated in the pathogenesis of systemic lupus erythematosus due to its role in regulating the expression of proinflammatory cytokines such as IFN-α, IL-6, TNF-α, and IL-12 and pathogenic autoantibody production (Song et al., 2020). In an unstimulated condition, IRF5 is generally localized in the cytoplasm as a monomer (Thompson et al., 2018). Activation of the above receptors triggers cellular signaling cascades. IRF5 undergoes post-translational modification, which eventually leads to homodimerization, a critical event prior to nuclear translocation (Thompson et al., 2018). Here, we describe two methods designed for detecting IRF5 molecular interactions.


A native-PAGE method is used to detect endogenous IRF5 homodimers. THP-1 cells are incubated with or without (1 and 10 μM) IRF5-CPP inhibitors for 30 min, and subsequently stimulated with 1 μM of R848 (a TLR7 ligand) or left unstimulated for 1 h. Next, cell lysates are run on native polyacrylamide gel electrophoresis (PAGE) and IRF5 dimerization is detected by immunoblotting with IRF5 and HRP-conjugated secondary antibodies. The intracellular fluorescence resonance energy transfer (FRET) assay is used to detect binding of endogenous IRF5 to FITC-IRF5-CPP conjugated inhibitors through a FRET signal (Banga et al., 2020; Song et al., 2020). FRET is a technique designed for detecting molecular interaction in which the excited molecule (the donor) transfers non-radiative energy to another molecule (the acceptor) within a distance of ~1-10 nm (Doucey et al., 2003; Ujlaky-Nagy et al., 2018; Banga et al., 2020). THP-1 cells are incubated with or without (1 and 10 μM) IRF5-CPP inhibitors for 1 h. The untreated and FITC-IRF5-CPP- treated THP-1 cells are then fixed, permeabilized, and stained with anti-IRF5, anti-IRF3, or anti-IRF7 (other IRF family members) antibodies and TRITC secondary antibodies. Cell-associated fluorescence is measured on a BioTek Synergy Neo2 at 525 nm upon excitation at 488 nm (E1), at 600 nm upon excitation at 540 nm (E2), and at 600 nm upon excitation at 488 nm (E3). The transfer of fluorescence is calculated as FRET units as follows: FRET unit = (E3both E3none) − ([E3TRITCE3none) × (E2both/E2TRITC]) − ([E3FITCE3none] × [E1both/E1FITC]) (Doucey et al., 2003; Banga et al., 2020; Song et al., 2020). The different fluorescence values (E) were measured on unlabeled cells (Enone) or cells labeled with FITC (EFITC) and TRITC (ETRITC) (Banga et al., 2020; Song et al., 2020).


These techniques can be broadly applied to evaluate intracellular molecular interactions in living cells through pharmacological and molecular studies. They provide a rapid and reliable method of screening molecular interactions, which require standard equipment that are readily available in almost every laboratory.


Part I. Native-PAGE (Polyacrylamide Gel Electrophoresis)


Materials and Reagents

  1. THP-1 cells (ATCC, catalog number: TIB-202)

  2. RPMI-1640 medium (Gibco, catalog number: 11875093)

  3. Fetal bovine serum (Gibco, catalog number: 16000044)

  4. Penicillin-streptomycin (Gibco by Life Technologies, catalog number: 15140122)

  5. IRF5 cell penetrating peptides (IRF5-CPP) and FITC-IRF5-CPP inhibitors (Hoffman-LaRoche, Patent number WO2014001229A2)

  6. Resiquimod (R848) (Millipore Sigma, catalog number: SML0196)

  7. Sodium deoxycholate (DOC) (Thermo Fisher, catalog number: 89904)

  8. DC protein assay (Bio-Rad, catalog number: 5000111)

  9. BCA protein assay (Thermo Fisher, catalog number: 23227)

  10. Anti-IRF3, rabbit monoclonal antibody (Abcam, catalog number: ab76409)

  11. Anti-IRF5 (Cell Signaling Tech, catalog number: 3257, rabbit polyclonal antibody or catalog number: 13496, rabbit monoclonal antibody)

  12. Anti-IRF7, rabbit polyclonal antibody (Cell Signaling Tech, catalog number: 4920)

  13. Horseradish peroxidase (HRP)–conjugated anti-β-actin, rabbit monoclonal antibody (Cell Signaling Tech, catalog number: 5125)

  14. Anti-rabbit IgG HRP-conjugated secondary antibody (Cell Signaling Tech, catalog number: 7074)

  15. NP40 cell lysis buffer (Invitrogen, catalog number: FNN0021)

  16. Phenylmethylsulfonyl fluoride (PMSF) (Millipore Sigma, catalog number: 10837091001)

  17. Protease inhibitor (Millipore Sigma, catalog number: P-2714)

  18. Native gel running buffer, Tris-glycine buffer 10× concentrate, pH 8.3 (Millipore Sigma, catalog number: T4904-1L)

  19. Glycerol (Millipore Sigma, catalog number: G9012)

  20. Immobilon-P 0.45 μm PVDF membrane (Millipore, catalog number: IPVH00010)

  21. Bovine serum albumin (BSA) (Millipore Sigma catalog number: 05470-5G)

  22. Nonfat-dried milk bovine (Millipore Sigma, catalog number: M7409-1BTL)

  23. Bromophenol blue (Millipore Sigma, catalog number: B5525)

  24. 30% acrylamide/Bis solution, 37.5:1, 500 ml (Bio-Rad, catalog number: 161-0158)

  25. Resolving gel buffer, 1.5 M Tris-HCl, pH 8.8, 1 L (Bio-Rad, catalog number: 161-0798)

  26. Stacking gel buffer, 0.5 M Tris-HCl, pH 6.8, 1 L (Bio-Rad, catalog number: 161-0799)

  27. SDS solution, 10% (w/v), 250 ml (Bio-Rad, catalog number: 161-0416)

  28. Pierce 20× TBS Tween 20 buffer (TBST) (Thermo Fisher, catalog number: 28360)

  29. Western bright ECL HRP substrate (Advansta, catalog number: K-12045-C20)

  30. Precision plus protein standard (Bio-Rad, catalog number: 161-0374) in which high molecular weight bands can be detected on native gel

Equipment

  1. ChemiDoc MP Imaging System (Bio-Rad, catalog number: 17001402)

  2. Mini-Transblot Cell and PowerPac Basic Power Supply (Bio-Rad, catalog number: 1703989)

  3. Mini-Protean Tetra Vertical Electrophoresis Cell for Mini Precast Gels (Bio-Rad, catalog number: 1658005)

Procedure



Figure 1. Overview of native-PAGE method designed for detecting endogenous IRF5 homodimers

  1. R848-induced IRF5 homodimerization and inhibition of IRF5 homodimerization by IRF5-CPPs in THP-1

    1. Plate THP-1 cells (6 × 106/ml) in complete RPMI-1640 medium in a 6-well plate and keep in the incubator (37°C, 5% CO2) for 24 h.

    2. Prepare 1 and 10 µM of IRF5-CPP and 1 µM of R848 in complete RPMI-1640 medium. Incubate THP-1 cells with or without final concentration of IRF5-CPP for 30 min. Subsequently add 1 µM of R848 and incubate at 37°C, 5% CO2 for 1 h.

    3. Transfer cells and media containing 6 × 106 THP-1 cells into a 5 ml or 15 ml tube.

    4. Centrifuge samples at 400 × g, 4°C for 5 min. Aspirate supernatant without disturbing the cell pellet.

    5. Wash cells with 5 ml PBS and centrifuge at 400 × g, 4°C for 5 min. Aspirate PBS without disturbing the cell pellet.


  2. Cell Lysis and Protein Concentration Measurement

    1. Prepare cell lysis buffer by adding 1 mM PMSF and 500 μl of 10× protease inhibitor cocktail to 5 ml NP40 lysis buffer immediately prior to use. (NP40 lysis buffer, instructions from the manufacturer: http://tools.thermofisher.com/content/sfs/manuals/FNN0021_Rev%200908.pdf)

    2. Lyse the cell pellet in NP40 cell lysis buffer for 30 min, on ice, and vortex every 10 min. Add 500 μl of NP40 cell lysis buffer to the cell pellet. (The volume of cell lysis buffer depends on the cell number and expression of target protein.)

    3. Transfer the extract to microcentrifuge tubes and centrifuge at 8,000 × g for 10 min at 4°C.

    4. Aliquot the clear lysate to clean microfuge tubes. These samples are ready for BCA or DC protein assay. Lysates can be stored at -80°C. Avoid multiple freeze/thaws.

    5. Measure the protein concentration using BCA or DC protein assay as per kit instruction. Keep samples on ice to prevent protein denaturation.


  3. 8% Native Polyacrylamide Gel Hand Casting

    Make 8% Native Gel (as per Bio-Rad Casting Instruction, http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110106B.pdf).

    Note: Substitute SDS with deionized distilled water. SDS is a known amphipathic surfactant that denatures proteins. Do NOT use SDS except for gel incubation post-native PAGE.


  4. Native-PAGE and Immunoblotting

    1. Put pre-running native running buffer (25 mM Tris and 192 mM glycine, pH 8.3) in the freezer until it reaches 0°C temperature. Make sure that there are no ice crystals prior to pouring into the electrophoresis chamber unit.

    2. Pre-run native gel with native running buffer with 1% DOC in the cathode chamber, and native running buffer in the anode chamber at 40 mA, 0°C for 30 min. Keep electrophoresis unit on ice and maintain 0°C temperature (Figure 2).

    3. Prepare samples for native gel electrophoresis by mixing the sample with native sample buffer. The final protein concentration per sample is 10 µg/well. Do NOT heat the samples.

      NOTE: Heating will denature the proteins which will lead to dissociation of IRF5 dimers.



      Figure 2. Schematic diagram of native-PAGE set up


    4. Add fresh cold native running buffers in the anode and cathode chambers (with 1% DOC in the cathode). Keep electrophoresis unit on ice and maintain at a temperature of 0°C (Figure 2).

    5. Load the ladder and samples (total protein concentration: 10 µg per sample per well).

    6. Electrophorese samples at 25 mA, 0°C, for 60 min or until you see the dye close to the bottom.

    7. Incubate gel in native running buffer containing 0.1% SDS for 30 min.

      NOTE: Dilute 10% SDS in deionized distilled water. The addition of 0.1% SDS is to improve transfer efficiency.

    8. Proceed to protein transfer on PVDF or nitrocellulose membrane.

    9. Pre-wet the membrane and place the gel/membrane sandwich into the transfer apparatus.

    10. Add fresh transfer buffer to the tank.

    11. Run at 100 V for 60-70 min while keeping the tank on ice.

    12. After transfer, air-dry the PVDF membrane.

    13. Briefly soak in 100% methanol to wet the membrane.

    14. Wash the membrane with distilled water 3× to remove the methanol.

    15. Block the membrane with 5% non-fat milk-TBST or 5% BSA-TBST solution for 1 h.

      NOTE: Dilute 20× TBST in distilled water to make 1× TBST.

    16. Wash the membrane 3× with TBST.

    17. Probe the membrane with primary antibody (IRF5, IRF3 or IRF7) on a rocker at 4°C overnight.

    18. Wash the membrane 3× with TBST.

    19. Probe the membrane with HRP-conjugated secondary antibody on a rocker at room temperature for 1 h.

    20. Wash the membrane 3× with TBST.

    21. Proceed with chemiluminescent detection of proteins using ChemiDoc Imaging System.


    The resulting immunoblot (Figure 3) shows that R848 treatment increased endogenous IRF5 dimer (IRF5)2 formation and that IRF5-CPP5 and not IRF5-CPP2 was able to inhibit dimerization by R848. The IRF5 monomer (IRF5) and β-actin (loading control) are shown.



    Figure 3. Native-PAGE. On lane 1 control – untreated THP-1, lane 2 treated with 1 μM R848, lane 3 treated with 1 μM IRF5-CPP2 and 1 μM R848, lane 4 treated with 10 μM IRF5-CPP2 and 1 μM R848, lane 5 treated with 1 μM IRF5-CPP5 and 1 μM R848, and lane 6 treated with 10 μM IRF5-CPP5 and 1 μM R848.

Recipes

  1. Native Sample Buffer

    To make 10 ml of Native Sample Buffer, mix 15% glycerol (v/v), 1% DOC (w/v), and 1.25 ml of 0.5 M Tris-Cl, pH 6.8 and 0.1% bromophenol blue (w/v) and deionized distilled water.

  2. Native Running Buffer

    To make 1 L of Native Running Buffer, mix 100 ml of 10× Tris-Glycine Buffer (pH 8.3) and 900 ml of deionized distilled water.

  3. Transfer Buffer

    To make 1 L of Transfer Buffer, add 100 ml of 10× transfer buffer, 100 ml of methanol and 800 ml of deionized distilled water.

  4. Complete RPMI-1640 Medium

    Mix the following: a bottle of 500 ml RPMI-1640, 10% FBS, 1% Penicillin-Streptomycin


Part II. In-cell Fluorescence Resonance Energy Transfer (FRET)

Materials and Reagents

  1. Corning 96-well black plates (Fisher Scientific, catalog number: 07200627)

  2. 5 ml macrotubes (Fisher Scientific, catalog number: 501537789)

  3. THP-1 cells (ATCC, catalog number: TIB-202)

  4. RPMI-1640 Medium (Gibco, catalog number: 11875093)

  5. Fetal Bovine Serum (Gibco, catalog number: 16000044)

  6. Penicillin-Streptomycin (Gibco by Life Technologies, catalog number: 15140122)

  7. FITC-IRF5-CPP inhibitors (Hoffman-LaRoche, Patent WO2014001229A2)

  8. Resiquimod (R848) (Millipore Sigma, catalog number: SML0196)

  9. Foxp3 Fixation/Permeabilization Buffer (Invitrogen, catalog number: 00-5523-00)

  10. Bovine serum albumin (BSA) (Millipore Sigma catalog number: 05470-5G)

  11. Permeabilization buffer (Invitrogen, catalog number: 00-5523-00)

  12. Anti-IRF5, rabbit monoclonal antibody (Abcam, catalog number: ab124792)

  13. Goat anti-rabbit tetramethyl rhodamine isothiocyanate (TRITC) antibody (Abcam, catalog number: ab6718)

Equipment

  1. Biotek Synergy Neo2 (Biotek, model: BTNEO2) or any similar plate reader

Procedure

  1. Buffer and Solution Preparation

    1. Prepare fresh Foxp3 Fixation/Permeabilization working solution by mixing 1 part of Foxp3 Fixation/Permeabilization concentrate with 3 parts of PBS containing 2% BSA. For 3-5 million cells, 1 ml of the working solution is required for each sample.

    2. Prepare 1× working solution of Permeabilization Buffer by mixing 1 part of 10× Permeabilization Buffer with 9 parts of deionized distilled water. 1 ml of the working solution is required for each sample.


  2. Detection of endogenous IRF5 interaction with FITC-IRF5-CPP inhibitors in THP-1

    1. Plate 2 × 105-4 × 105/ml THP-1 cells in complete RPMI-1640 media in a 96-well plate (in triplicate per condition/treatment) and keep in the incubator (37°C, 5% CO2) for 24 h.

    2. Prepare 1 and 10 µM of FITC-IRF5-CPP conjugated inhibitors in complete RPMI-1640 medium. Aspirate medium and incubate THP-1 cells with or without FITC-IRF5-CPP in 37°C, 5% CO2 for 1 h.

      NOTE: Keep the samples protected from light.

    3. Transfer the FITC-IRF5-CPP treated cells in to a 5 ml tube.

    4. Centrifuge at 400 × g, 4°C for 5 min. Aspirate supernatant without disturbing the cell pellet.

    5. Wash cells with 3 ml PBS and centrifuge at 400 × g, 4°C for 5 min. Aspirate PBS without disturbing the cell pellet.

    6. Add 1 ml of Foxp3 Fixation/Permeabilization working solution to each tube and pulse vortex.

    7. Incubate for 30-60 min at room temperature or for up 18 h at 2-8°C.

    8. Cells should have settled in the bottom after 18 h. Carefully aspirate supernatant. For short incubation (30-60 min at room temperature), spin at 400 × g, 4°C for 5 min.

    9. Add 1 ml of 1× Permeabilization Buffer to each tube and incubate for 30 min at room temperature.

    10. Spin at 400 × g for 5 min.

    11. Resuspend pellet in 50 µl residual volume of 1× Permeabilization Buffer.

    12. [Optional] Block with 10% BSA by adding 5 µl directly to each tube. Incubate for 15 min at room temperature.

    13. Without washing, add the recommended amount of primary (anti-IRF5) antibody for detection of intracellular antigen and incubate for at least 30 min at room temperature. Protect from the light.

    14. Wash with 3 ml of PBS.

    15. Repeat Steps B9-B12 and add the recommended amount of secondary (TRITC) antibody (to bind to anti-IRF5 antibody).

    16. Spin at 400 × g for 5 min. Aspirate supernatant leaving 30 µl residual volume.

    17. Add 30 µl of 4% PFA and resuspend stained cells. Protect from the light.

    18. Transfer samples to a 96-well plate. Prior to reading, add PBS to a final volume of 150-200 µl.

      NOTE: Make sure you have the same number of cells per well. Counting the cells is highly recommended.

    19. Measure intracellular fluorescence on Biotek Synergy Neo2 (Bioteck, VT, USA) or any similar plate reader at 525 nm upon excitation at 488 nm (E1), at 600 nm after excitation at 540 nm (E2), and 600 nm after excitation at 488 nm (E3).

      Set up the machine with 3 spectra:

      FITC  488 excitation 525 emission

      TRITC  540 excitation 600 emission

      FRET  488 excitation 600 emission

    20. The transfer of fluorescence was calculated as FRET units as follows: FRET unit = (E3c − E3none) − ([E3TRITC − E3none) × (E2none/E2TRITC]) − ([E3 FITC −E3none] × [E1both/E1TRITC ]). The different fluorescence values (E) were measured on unlabeled cells (Enone) or cells labeled with FITC (EFITC) and TRITC (ETRITC).


    The resulting bar graph (Figure 4) shows an increase in FRET signal (unit) between endogenous IRF5 and FITC-IRF5-CPP2 or FITC-IRF5-CPP5 but not FITC-IRF5-CPP8 or –CPP9.



    Figure 4. Binding of IRF5-CPPs to IRF5. THP-1 cells were incubated with FITC-IRF5-CPP2, FITC-IRF5-CPP5, FITC-IRF5-CPP8, or FITC-IRF5-CPP9 for 1 h, followed by permeabilization and staining for intracellular IRF5 with anti-IRF5 and tetramethyl rhodamine isothiocyanate (TRITC) antibodies. FRET units were calculated from fluorescence emissions.

Acknowledgments

Funding: This work was supported and funded by F. Hoffmann–La Roche, the Lupus Research Alliance (to B.J.B.), Department of Defense CDMRP Lupus Research Program W81XWH-18-1-0674 (to B.J.B.), NIH AR065959 (to B.J.B.), and EMD Serono Research and Development Institute Inc.

Competing interests

Disclosures: J.A.D., S.-L.T., and D.S. are inventors on patent application US20160009772A1 assigned to F. Hoffmann–La Roche AG. Application status abandoned as of 12 May 2019 as a matter of public record. Financial disclosures related to companies: G.C., C.-C.S., J.Q., M.D., and J.A.D. are employees of EMD Serono Research and Development Institute Inc. S.H. is employee of BMS. F.M. is the author of patent “Cell penetrating peptides & methods of identifying cell penetrating peptides” (WO2014001229A2) filed by F. Hoffmann–La Roche. J.A.D., N.F., A.F.H., K.-S.H., F.M., D.S., and S.-L.T. are authors of patent “Cell penetrating peptides which bind IRF5” (US20160009772A1) filed by Hoffmann–La Roche Inc.

References

  1. Banga, J., Srinivasan, D., Sun, C. C., Thompson, C. D., Milletti, F., Huang, K. S., Hamilton, S., Song, S., Hoffman, A. F., Qin, Y. G., Matta, B., LaPan, M., Guo, Q., Lu, G., Li, D., Qian, H., Bolin, D. R., Liang, L., Wartchow, C., Qiu, J., Downing, M., Narula, S., Fotouhi, N., DeMartino, J. A., Tan, S. L., Chen, G. and Barnes, B. J. (2020). Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.Sci Adv 6(20): eaay1057.
  2. De, S., Zhang, B., Shih, T., Singh, S., Winkler, A., Donnelly, R. and Barnes, B. J. (2017). B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation. Front Immunol 8: 1938.
  3. Doucey, M. A., Goffin, L., Naeher, D., Michielin, O., Baumgartner, P., Guillaume, P., Palmer, E. and Luescher, I. F. (2003). CD3 delta establishes a functional link between the T cell receptor and CD8. J Biol Chem 278(5): 3257-3264.
  4. Song, S., De, S., Nelson, V., Chopra, S., LaPan, M., Kampta, K., Sun, S., He, M., Thompson, C. D., Li, D., Shih, T., Tan, N., Al-Abed, Y., Capitle, E., Aranow, C., Mackay, M., Clapp, W. L. and Barnes, B. J. (2020). Inhibition of IRF5 hyperactivation protects from lupus onset and severity. J Clin Invest 130(12): 6700-6717.
  5. Thompson, C. D., Matta, B. and Barnes, B. J. (2018). Therapeutic Targeting of IRFs: Pathway-Dependence or Structure-Based? Front Immunol 9: 2622.
  6. Ujlaky-Nagy, L., Nagy, P., Szollosi, J. and Vereb, G. (2018). Flow Cytometric FRET Analysis of Protein Interactions. Methods Mol Biol 1678: 393-419.

简介

[摘要]干扰素调节因子5(IRF5)二聚化测定细胞内是设计一种技术,以测量电È分子相互作用(S)与内源性IRF5。在这里,我们介绍了两种检测内源性IRF5同源二聚化和内源性IR5与细胞穿透肽(CPP)抑制剂相互作用的方法。简要地说,以检测内源IRF5二聚体,THP-1细胞是在存在或不存在下温育的IRF5靶向CPP(IRF5-CPP)抑制剂30分钟,然后将细胞刺激与R848 1个小时。细胞裂解物是 通过使用IRF5抗体进行免疫印迹,可以检测到通过天然聚丙烯酰胺凝胶电泳(PAGE)和IRF5二聚体分离的产物。为了检测内源之间的相互作用IRF5和FITC标记IRF5-CPP,一个在细胞荧光共振能量转移(FRET)测定我š使用。在该测定中,THP-1细胞一个重新左未处理或处理用FITC- IRF5-CPP缀合抑制剂1个小时。接着,C ELLS一个重新固定,透化,并用抗IRF5染色和TRITC标记的二抗。可以测量荧光的转移并将其计算为FRET单位。这些方法提供了快速,准确的检测IRF5分子相互作用的检测方法。


[背景]干扰素调节因子5(IRF5)是一种转录因子,可调节病原体诱导的Toll样受体(TLR),视黄酸诱导型基因I(RIG-I)下游,黑素瘤分化相关的先天和后天免疫应答。基因5(MDA5)和B细胞受体(BCR)(De等,2017; Thompson等,2018; Banga等,2020 )。IRF5已牵涉于系统性红斑狼疮的发病机理是由于其在调节促炎细胞因子如IFN-α,IL-6,TNF-α和IL-12和致病的自身抗体生产的表达的作用(宋等人, 2020年)。在不受刺激的条件下,IRF5通常作为单体定位在细胞质中(Thompson等,2018 )。上述受体的激活触发细胞信号级联反应。IRF5经过翻译后修饰,最终导致同源二聚化,这是核易位之前的关键事件(Thompson等,2018 )。在这里,我们描述了两种用于检测IRF5分子相互作用的方法。

甲天然-PAGE方法我š用于检测内源性IRF5同二聚体。THP-1细胞在有或没有(1和10 μ中号)IRF5-CPP抑制剂30分钟,随后用1刺激μ中号R848的(一个TLR7配体)或没有刺激1个小时。接着,C ELL裂解物一重新上天然聚丙烯酰胺凝胶电泳(PAGE)和IRF5二聚化运行我S按与IRF5和HRP缀合的二级抗体进行免疫印迹检测。细胞内荧光共振能量转移(FRET)测定我š用于检测内源性IRF5到FITC-IRF5-CPP的结合通过FRET信号缀合抑制剂(邦加等人,2020;宋等人,2020 )。FRET是一种设计用于检测分子相互作用的技术,其中受激发的分子(供体)在约1至10 nm的距离内将非辐射能转移至另一个分子(受体)(Doucey等,2003; Ujlaky-Nagy等人,2018; Banga等人,2020 )。THP-1细胞一个再有或没有(1和10孵育μ中号)IRF5-CPP抑制剂1个小时。未处理的和FITC-IRF5-CPP-处理的THP-1细胞中一个重新然后固定,透化,并用抗IRF5,抗IRF3,或抗IRF7(其他IRF家庭成员)的抗体染色和TRITC次级抗体。细胞相关的荧光我S于测量BioTek的协同NEO2在时在488nm(E1)激发525纳米,在540nm处(E2)激发为600nm,并且在在488nm(E3)激发后600纳米。荧光的转移我S作为如下计算为FRET单元:FRET单元=(ë 3两者- ë 3无) - ([ ë 3 TRITC - ë 3无)×(É 2二者/ ë 2 TRITC ]) - ([ E 3 FITC - E 3 none ]×[ E 1 both / E 1 FITC ])(Doucey等,2003;Banga等,2020;Song等,2020 )。在未标记的细胞(E none )或用FITC(E FITC )和TRITC(E TRITC )标记的细胞上测量了不同的荧光值(E )(Banga等,2020; Song等,2020 )。

这些技术可以广泛地用于通过药理和分子研究来评估活细胞中的细胞内分子相互作用。它们提供了一种快速可靠的筛选分子相互作用的方法,这需要几乎在每个实验室中都可以买到的标准设备。

关键字:细胞内干扰素调节因子5, 非变性聚丙烯酰胺凝胶电泳, 聚丙烯酰胺凝胶电泳, 荧光共振能量转移, 二聚作用, 分子间相互作用, 干扰素调节因子5-细胞穿透肽


部分一,本机-PAGE(聚丙烯酰胺凝胶电泳)


材料和试剂


THP-1细胞(ATCC ,目录号:TIB-202)
RPMI-1640米edium(GIBCO,目录号:11875093)
胎b绵羊小号erum(GIBCO,目录号:16000044)
青霉素小号treptomycin(由Life Technologies公司GIBCO,目录号:15140122)
IRF5细胞穿透肽(IRF5-CPP)和FITC-IRF5-CPP抑制剂(Hoffman-LaRoche,专利号WO2014001229A2)
Resiquimod (R848)(Millipore Sigma,目录号:SML0196)
脱氧胆酸钠(DOC)(Thermo Fisher,目录号:89904)
DC p rotein一个SSAY(Bio-Rad公司,目录号:5000111)
BCA p rotein一个SSAY(赛默飞世,目录号:23227)
抗IRF3,兔单克隆抗体(Abcam,目录号:ab76409)
抗IRF5(Cell Signaling Tech,目录号:3257,兔多克隆抗体或目录号:13496,兔单克隆抗体)
抗IRF7,兔多克隆抗体(Cell Signaling Tech,目录号:4920)
辣根过氧化物酶(HRP)缀合的抗- β肌动蛋白,兔单克隆抗体(Cell Signaling技术,目录号:5125)
抗兔IgG HRP共轭二抗(Cell Signaling Tech,目录号:7074)
NP40 Ç ELL升ysis b uffer(Invitrogen公司,目录号:FNN0021)
苯甲基磺酰氟(PMSF)(Millipore Sigma,目录号:10837091001)
蛋白酶我nhibitor器(Millipore西格玛,目录号:P-2714)
天然克EL ř unning b uffer,三-克甜菜碱b uffer 10 × Ç oncentrate,pH值8.3(Millipore公司Sigma公司,目录号:T4904-1L)
甘油(Millipore Sigma,目录号:G9012)
的Immobilon -P 0.45微米PVDF膜(Millipore,目录号:IPVH00010)
牛小号erum一个lbumin(BSA)(Sigma公司的Millipore目录号:05470-5G)
Nonfat- d RIED米之流b绵羊(Millipore公司Sigma公司,目录号:M7409-1BTL)
溴酚蓝(Millipore Sigma,目录号:B5525)
30%一crylamide /双小号olution,37.5:1,将500ml(Bio-Rad公司,目录号:161-0158)
解决克EL b uffer,1.5M的Tris-HCl,pH值8.8,1 L(Bio-Rad公司,目录号:161-0798)
堆叠克EL b uffer,0.5的1M Tris-HCL,pH为6.8,1 L(Bio-Rad公司,目录号:161-0799)
SDS小号olution,10%(W / V)加入250ml(Bio-Rad公司,目录号:161-0416)
皮尔斯20 × TBS吐温20 b uffer(TBST)(赛默飞世,目录号:28360)
西方b右ECL HRP小号ubstrate(Advansta ,目录号:K-12045-C20)
精度p的LU p rotein小号TANDARD(Bio-Rad公司,目录号:161-0374),其中高分子量条带可以在天然凝胶来检测


设备


ChemiDoc MP成像系统(Bio-Rad,目录号:17001402 )
Mini- Transblot电池和PowerPac基本电源(Bio-Rad,目录号:1703989)
用于迷你预制凝胶的Mini-Protean Tetra垂直电泳池(Bio-Rad,目录号:1658005)


程序




图1概述设计天然-PAGE法对d etecting Ë ndogenous IRF5同型二聚体


R848诱导的IRF5同源二聚化和THP-1中IRF5-CPP抑制IRF5同源二聚化
将THP-1细胞(6 × 10 6 / ml)置于6孔板的完全RPMI-1640培养基中,并在培养箱(37°C,5%CO 2 )中放置24小时。
在完全RPMI-1640培养基中制备1和10 µM的IRF5-CPP以及1 µM的R848。将THP-1细胞与最终浓度的IRF5-CPP一起或不与之孵育30分钟。因此,添加1 µM R848,并在37°C,5%CO 2下孵育1小时。
转移的细胞和含有6媒体× 10 6 THP-1细胞到一个5毫升或15米升管。
在400 × g ,4°C下将样品离心5分钟。吸出上清液而不干扰细胞沉淀。
用5 ml PBS洗涤细胞,并在400 × g ,4°C下离心5分钟。吸出PBS而不干扰细胞沉淀。


细胞裂解和蛋白质浓度测量
通过加入制备细胞裂解缓冲液1mM的PMSF和500 μ升10 ×蛋白酶抑制剂混合物至5μm升NP40裂解缓冲液在使用前立即。(NP40裂解缓冲液,从所述指令制造商:http://tools.thermofisher.com/content/sfs/manuals/FNN0021_Rev%200908.pdf)
在冰上在NP40细胞裂解缓冲液中裂解细胞沉淀30分钟,然后每10分钟涡旋一次。加入500 μ升NP40的细胞裂解缓冲液的细胞沉淀。(细胞裂解缓冲液的量取决于细胞数量和靶蛋白的表达。)
转移提取物离心管并离心在8 ,000 ×克在4 10分钟℃。
分装澄清的裂解液以清洁微量离心管。这些样品已准备好进行BCA或DC蛋白测定。裂解液可在-80°C下保存。避免多次冻结/解冻。
按照试剂盒说明使用BCA或DC蛋白质测定法测量蛋白质浓度。将样品放在冰上以防止蛋白质变性。


8%非变性聚丙烯酰胺凝胶手CASTIN克
制作8%天然凝胶(按照Bio-Rad铸造说明,http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110106B.pdf)。


注:替换SDS与去离子蒸水。SD S是已知的使蛋白质变性的两亲性表面活性剂。除天然PAGE后的凝胶孵育外,请勿使用SDS。


天然PAGE和免疫印迹
将预先运行的本机运行缓冲液(25 mM Tris和192 mM甘氨酸,pH 8.3)放入冰箱,直至达到0°C温度。倒入电泳室之前,请确保没有冰晶。
预运行天然凝胶,在40 mA,0°C下,在阴极室中使用1%DOC的自然运行缓冲液,在阳极室中使用自然运行缓冲液30分钟。保持在冰上电泳单元和保持0℃的温度(图URE 2 )。
通过将样品与天然样品缓冲液混合,准备用于天然凝胶电泳的样品。每个样品的最终蛋白质浓度为10 µg /孔。不要加热样品。
注:^ h进食会变性,这将导致IRF5二聚体分解蛋白质。




图URE 2 。本机页面设置的示意图


在阳极室和阴极室中添加新鲜的自然冷运行缓冲液(阴极中含1%DOC)。保持在冰上电泳单元和保持在温度0℃(图URE 2 )。
装载梯子和样品(总蛋白浓度:每个样品每孔10 µg)。
在25 mA,0°C下电泳60分钟或直到您看到染料靠近底部为止。
在含有0.1%SDS的天然运行缓冲液中孵育凝胶30分钟。
注:d ilute 10%SDS在去离子蒸馏水水。添加0.1%SDS可以提高传输效率。


进行蛋白质在PVDF或硝酸纤维素膜上的转移。
预湿膜,然后将凝胶/膜三明治放入转移设备中。
将新鲜的转移缓冲液添加到水箱中。
在将水箱置于冰上的同时,在100 V下运行60-70分钟。
转移后,风干PVDF膜。
短暂地浸泡在100%甲醇中以润湿膜。
用蒸馏水洗涤膜3倍,以除去甲醇。
用5%脱脂牛奶-TBST或5%BSA-TBST溶液封闭膜1小时。
注意:在蒸馏水中稀释20 × TBST,制成1 × TBST。


洗膜3 ×用TBST。
在摇杆上于4 °C过夜用一抗(IRF5,IRF3或IRF7)探测膜。
洗膜3 ×用TBST。
在室温下在摇床上用HRP偶联的二抗探测膜1小时。
洗膜3 ×用TBST。
继续使用ChemiDoc Imaging System对蛋白质进行化学发光检测。


将得到的免疫印迹(图URE 3 )表明,R848处理增加内源性IRF5二聚体(IRF5)2的形成和IRF5-CPP5和不IRF5-CPP2能够由R848抑制二聚化。显示了IRF5单体(IRF5)和β-肌动蛋白(上样对照)。




图URE 3 。本机页面。泳道1控制-未处理的THP-1,用1处理泳道2 μ中号R848,泳道3用1处理μ中号IRF5-CPP2和1 μ中号R848,泳道4用10处理μ中号IRF5-CPP2和1 μ中号R848 ,泳道5 1处理μ中号IRF5-CPP5和1 μ中号R848,和泳道6,用10处理μ中号IRF5-CPP5和1 μ中号R848。


菜谱


本机样本缓冲区
要制备10 ml的天然样品缓冲液,请混合15%甘油(v / v),1%DOC(w / v)和1.25 ml 0.5 M Tris-Cl,pH 6.8和0.1%溴酚蓝(w / v)和去离子蒸馏水。


              本机运行缓冲区
要制成1 L的本机运行缓冲液,请混合100 ml的10 × Tris-甘氨酸缓冲液(pH 8.3)和900 ml去离子蒸馏水。


传输缓冲区
要制成1 L的转移缓冲液,请添加100 ml的10 ×转移缓冲液,100 ml的甲醇和800 ml的去离子蒸馏水。


完整的RPMI-1640中
混合以下内容:一瓶500毫升RPMI-1640、10%FBS,1%青霉素-链霉素


部分II。细胞内荧光共振能量转移(FRET)


材料和试剂


康宁96孔黑色板(Fisher Scientific,目录号:07200627)
5毫升宏管小号(费舍尔科学,目录号:501537789)
THP-1细胞(ATCC ,目录号:TIB-202)
RPMI-1640中等(Gibco,目录号:11875093)
胎牛血清(Gibco,目录号:16000044)
青霉素-链霉素(Gibco by Life Technologies,目录号:15140122)
FITC-IRF5-CPP抑制剂(Hoffman-LaRoche,专利WO2014001229A2)
Resiquimod (R848)(Millipore Sigma,目录号:SML0196)
Foxp3固定/通透缓冲液(Invitrogen,目录号:00-5523-00)
牛小号ERU米一个lbumin(BSA)(Sigma公司的Millipore目录号:05470-5G)
透b uffer (Invitrogen公司,目录号:00-5523-00)
抗IRF5,兔单克隆抗体(Abcam,目录号:ab124792)
山羊抗兔四甲基罗丹明异硫氰酸酯(TRITC)抗体(Abcam,目录号:ab6718)


设备


Biotek Synergy Neo2(Biotek ,型号:BTNEO2)或任何类似的读板器


程序


缓冲液和溶液制备
通过将1份Foxp3固定/透化浓缩液与3份含2%BSA的PBS混合来制备新鲜的Foxp3固定/透化工作液。对于3-5百万个细胞,每个样品需要1 ml的工作溶液。
通过将1份10 ×透化缓冲液与9份去离子蒸馏水混合,制备1 ×透化缓冲液工作溶液。每个样品需要1 ml工作溶液。


检测THP-1中内源性IRF5与FITC-IRF5-CPP抑制剂的相互作用
在完整的RPMI-1640培养基中,将2 × 10 5 -4 × 10 5 / ml THP-1细胞接种在96孔板中(每个条件/处理一式三份)并保存在培养箱中(37°C,5%CO 2 )24小时。
在完整的RPMI-1640培养基中制备1和10 µM FITC-IRF5-CPP结合的抑制剂。吸出培养基,将含有或不含有FITC-IRF5-CPP的THP-1细胞在37°C,5%CO 2中孵育1小时。
注:ķ EEP样品保护避光。


将FITC-IRF5-CPP处理过的细胞转移到5 ml试管中。
在400 ×g ,4°C下离心5分钟。吸出上清液而不干扰细胞沉淀。
用3 ml PBS洗涤细胞,并在400 ×g ,4°C下离心5分钟。吸出PBS而不干扰细胞沉淀。
向每个试管和脉冲涡流中加入1 ml Foxp3固定/通透性工作溶液。
在室温下孵育30-60分钟,或在2-8°C下孵育18小时。
细胞应在18小时后沉淀在底部。小心吸出上清液。对于短暂的孵育(室温下30-60分钟),在400 ×g ,4°C下旋转5分钟。
向每个试管中加入1 ml的1 ×透化缓冲液,并在室温下孵育30分钟。
以400 ×g旋转5分钟。
将沉淀重悬于50 µl剩余体积的1 ×透化缓冲液中。
[可选]通过将5 µl直接加到每个试管中,用10%BSA封闭。在室温下孵育15分钟。
无需洗涤,添加推荐量的一抗(IRF5)抗体以检测细胞内抗原,并在室温下孵育至少30分钟。避光。
用3 ml PBS洗涤。
重复S teps B 9- B 12并添加推荐量的第二抗体(TRITC)(以结合抗IRF5抗体)。 
以400 ×g旋转5分钟。吸出上清液,剩余30 µl残留量。
加入30 µl 4%PFA,重悬染色的细胞。避光。
将样品转移到96孔板中。读取之前,添加PBS的终体积为150-200 µl。
注意:确保每个孔中的细胞数相同。强烈建议对细胞进行计数。


在488 nm(E1)激发下在525 nm,540 nm(E2)激发后在600 nm和488激发后600 nm在Biotek Synergy Neo2(Bioteck ,VT,USA)或任何类似的读板器上测量细胞内荧光nm(E3)。
用3个光谱设置机器:


FITC      488激发525发射           

TRITC    540激发600发射           

FRET     488激发600发射           

荧光的转移以FRET单位计算如下:FRET单位=(E 3 c - E 3无)-([ E 3 TRITC - E 3无)×(E 2无/ E 2 TRITC ])-([ E 3 FITC - E 3无]×[ E 1均为/ E 1 TRITC ] )。在未标记的细胞(E none )或用FITC(E FITC )和TRITC(E TRITC )标记的细胞上测量了不同的荧光值(E )。


将得到的柱状图(图URE 4 )示出了内源性IRF5和FITC- IRF5-CPP2或FITC-IRF5-CPP5但不是FITC-IRF5-CPP8或-CPP9之间的FRET信号(单元)的增加。




图4 。IRF5-CPP与IRF5的结合。将THP-1细胞与FITC-IRF5-CPP2,FITC-IRF5-CPP5,FITC-IRF5-CPP8或FITC-IRF5-CPP9孵育1小时,然后透化并用抗IRF5和四甲基罗丹明染色细胞内IRF5异硫氰酸盐(TRITC)抗体。从荧光发射计算出FRET单位。


致谢


资金:这项工作得到了F. Hoffmann–La Roche的支持和资助,狼疮研究联盟(对BJB),国防部CDMRP狼疮研究计划W81XWH-18-1-0674(对BJB),NIH AR065959(对BJB) ,以及EMD Serono研究与发展研究所有限公司。


利益争夺


披露:JAD,S.-LT和DS是转让给F. Hoffmann-La Roche AG的专利申请US20160009772A1的发明人。公开记录已于2019年5月12日终止了申请状态。与公司有关的财务披露:GC,C.-CS,JQ,MD和JAD是EMD Serono Research and Development Institute Inc.的雇员。SH是BMS的雇员。FM是F. Hoffmann–La Roche申请的专利“细胞穿透肽和鉴定细胞穿透肽的方法”(WO2014001229A2)的作者。JAD,NF,AFH,K.-SH,FM,DS和S.-LT是Hoffmann–La Roche Inc.申请的专利“结合IRF5的细胞穿透肽”(US20160009772A1)的作者。


参考


Banga,J.,Srinivasan,D.,Sun,CC,Thompson,CD,Milletti ,F.,Huang,KS,Hamilton,S.,Song,S.,Hoffman,AF,Qin,YG,Matta,B.,LaPan ,M.,Guo,Q.,Lu,G.,Li,D.,Qian,H.,Bolin,DR,Liang,L.,Wartchow,C.,Qiu,J.,Downing,M.,Narula ,S.,Fotouhi,N.,DeMartino,JA,Tan,SL,Chen,G。和Barnes,BJ(2020)。靶向同型二聚体的穿透细胞的肽对IRF5细胞活性的抑制作用。Sci Adv 6(20):eaay1057。
De,S.,Zhang,B.,Shih,T.,Singh,S.,Winkler,A.,Donnelly,R.和Barnes,BJ(2017)。IRF5在TLR9 / BCR诱导的人类B细胞活化,增殖和成浆细胞分化中的B细胞内在作用。免疫前线8:1938。
Doucey ,MA,Goffin,L.,Naeher,D.,Michielin ,O.,Baumgartner,P.,Guillaume,P.,Palmer,E。和Luescher,IF(2003)。CD3δ在T细胞受体和CD8之间建立功能连接。生物化学杂志278(5):3257-3264。
Song,S.,De,S.,Nelson,V.,Chopra,S.,LaPan ,M.,Kampta ,K.,Sun,S.,He,M.,Thompson,CD,Li,D.,Shih ,T.,Tan,N.,Al-Abed,Y.,Capitle ,E.,Aranow ,C.,Mackay,M.,Clapp,WL和Barnes,BJ(2020)。抑制IRF5过度活化可预防狼疮发作和严重程度。J临床投资130(12):6700-6717。
CD的汤普森,马塔·B和巴恩斯·巴恩斯(2018)。IRF的治疗目标:路径依赖还是基于结构?免疫学杂志9:2622。
Ujlaky-Nagy,L.,Nagy,P.,Szollosi,J.和Vereb,G.(2018)。蛋白质相互作用的流式细胞术FRET分析。方法分子生物学1678:393-419。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sherman, C. D. and Barnes, B. J. (2021). Intracellular IRF5 Dimerization Assay. Bio-protocol 11(10): e4021. DOI: 10.21769/BioProtoc.4021.
  2. Banga, J., Srinivasan, D., Sun, C. C., Thompson, C. D., Milletti, F., Huang, K. S., Hamilton, S., Song, S., Hoffman, A. F., Qin, Y. G., Matta, B., LaPan, M., Guo, Q., Lu, G., Li, D., Qian, H., Bolin, D. R., Liang, L., Wartchow, C., Qiu, J., Downing, M., Narula, S., Fotouhi, N., DeMartino, J. A., Tan, S. L., Chen, G. and Barnes, B. J. (2020). Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.Sci Adv 6(20): eaay1057.
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