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Dec 2017
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Hypoxia Reporter Element Assay
低氧反应元件的检测分析   

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Abstract

Hypoxia is a condition in which there is a decrease in oxygen supply to the cellular environment. Changes to cellular oxygen levels can lead to transcriptional changes of oxygen-regulated genes. Reporter assays are used to study gene expression alteration and modifications in response to environmental changes. Dual-reporter assays allow the simultaneous measurement of two different genes within a single cell, thus improving experimental accuracy. Within this protocol, we describe the utilization of the LightSwitch Dual Assay System to measure BMX expression in response to hypoxic conditions.

Keywords: Luciferase (荧光素酶), Reporter assay (报告系统检测技术), Hypoxia (低氧), Regulation (调控), Transcription (转录)

Background

In our recent publication (van Oosterwijk et al., 2018), we sought to examine the regulation of BMX, a nonreceptor tyrosine kinase, in response to sorafenib treatment. BMX, a Tec kinase family member, is known bind to tyrosine-phosphorylated proteins and mediate membrane targeting by binding to phosphatidylinositol 3,4,5-triphosphate (PIP3; Chen et al., 2013). We showed that direct treatment of sorafenib in acute myeloid leukemia (AML) cells lines with or without stromal cell support did not contribute to the upregulation of BMX. Previous studies have shown that BMX expression can be induced by ischemia and that sorafenib has antiangiogenic activity (He et al., 2006; Davis et al., 2011). Therefore, we hypothesized that the antiangiogenic activity of sorafenib causes a hypoxic environment within the bone marrow, thus contributing to a hypoxia-dependent BMX upregulation in AML. Under hypoxic conditions, we were able to show a significant increase in BMX expression in a number of different cell lines. Further analysis of the BMX promoter identified a putative hypoxia-responsive element (HRE; 5’-ACGTG-3’) at -5005. To test whether this HRE was involved in the hypoxia-induced promoter activation of BMX, we developed a hypoxia element reporter assay.

Reporter assays are extensively used throughout the scientific community to study alterations and modifications to gene expression in response to environmental changes. One type of reporter assay that is gaining in acceptance are the dual-reporter assays. This type of reporter assay allows the simultaneous expression and measurement of two different reporters within a single cell and had been widely proven to improve experimental accuracy by reducing extraneous influences. One such dual assay system is the LightSwitch Dual Assay System. This system utilizes the RenSP luciferase reporter gene, a novel luciferase developed by SwitchGear Genomics and the Cypridina luciferase reporter gene. RenSP and Cypridina employ different substrates, thus eliminating cross-reaction between proteins and their substrates. RenSP is used with your favorite gene as the reporter signal, and Cypridina is utilized with a constitutively active promoter as the control signal.

Here, we used the LightSwitch Dual Assay System to evaluate the involvement of the BMX HRE to its upregulation in response to hypoxic conditions.

Materials and Reagents

  1. Pipette tips
  2. White 96 well plates (CELLSTAR® Tissue Culture Plates, Greiner Bio One International, catalog number: 655083 )
  3. HEK293 (293[HEK-293]; ATCC, catalog number: CRL-1573 )
  4. pLightswitch_Prom_BMX (BMX promoter construct; SwitchGear Genomics, catalog number: S701154 )
  5. pLightswitch_Prom control vector (SwitchGear Genomics, catalog number: S790005 )
  6. pTK-Cluc (Cypridina TK control construct; SwitchGear Genomics, catalog number: SN0322S )
  7. FuGENE® HD (Promega, catalog number: E2311 )
  8. DMEM, high glucose, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11995065 )
  9. QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies, catalog number: 200516 )
  10. Terrific Broth Powder (BD, DifcoTM, catalog number: 243820 )
  11. Glycerol (Sigma-Aldrich, catalog number: G9012 )
  12. Poly-D-Lysine (Sigma-Aldrich, catalog number: P6407-5MG )
  13. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  14. Fetal Bovine Serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  15. LightSwitch Dual Assay System (SwitchGear Genomics, catalog number: DA010 )
  16. Poly-D-Lysine hydrobromide (MP Biomedicals, catalog number: 02150175 )
  17. Terrific Broth (see Recipes)
  18. 5x Poly-D-lysine Solution (see Recipes)
  19. 1x Poly-D-lysine Solution (sterile) (see Recipes)
  20. Growth media (see Recipes)

Equipment

  1. Pipettes
  2. Thermocycler (Eppendorf, model: Mastercycler® pro S , catalog number: 950030020)
  3. Table-top centrifuge (Eppendorf, model: 5810 R , catalog number: 022625101)
  4. Swinging bucket rotor (Eppendorf, model: S-4-104 , catalog number: 5820755008)
  5. High-performance centrifuge (Thermo Fisher Scientific, model: SorvallTM RC 6 Plus , catalog number: 36-101-0816)
  6. Laminar flow hood (Thermo Fisher Scientific, model: 1300 Series Class II , Type A2, catalog number: 1323TS)
  7. 37 °C, 5% CO2 water-jacketed incubator (Thermo Fisher Scientific, model: Heracell VIOS 160i , catalog number: 51030285)
  8. 37 °C non-humidified incubator (Labnet International, model: 311DS , catalog number: I5311-DS)
  9. Hypoxia chamber (Coy Lab, model: O2 Control InVitro Glove Box with no upgrades)
    Standard features include:
    1. Humidified incubation box
    2. Temperature control
    3. Control of O2 and CO2 in 0.1% increments
  10. Plate luminometer (Molecular Devices, model: SpectraMax i3x , with no upgrades)
    Standard features include:
    1. Read modes: Absorbance, Fluorescence, Luminescence
    2. Wavelength ranges include: Abs: 230-1,000 nm, Fl Ex: 250-830 nm, Fl EM 270-850 nm, Lumi: 300-850 nm

Software

  1. GraphPad Prism (GraphPad Software, Version 6)
  2. QuikChange Primer Design (https://www.genomics.agilent.com/primerDesignProgram.jsp)

Procedure

  1. Site-directed mutagenesis of pLightswitch_Prom_BMX to mutate the putative HRE within in the BMX promoter
    1. Primers are designed to mutate 2 base pairs (ACGTG → ATATG) within the HRE of the BMX promoter using the QuikChange Primer Design website (referenced above).
      The CG → TA bp change has been described in the literature to effectively disrupt the HRE.
    2. pLightSwitch_Prom_BMX is mutated using the QuikChangeXL Site-Directed Mutagenesis kit per manufacturer’s protocol.
      1. Extension time: 4.5 min
      2. Substitute DifcoTM Terrific Broth for NZY+ broth
    3. Mutated plasmids are confirmed by sequencing using promoter insert sequencing primers (Forward: 5’-TCCATCAAAACAAAACGAAACAA-3’, Reverse: 5’-AGTCGAGCACGTTCATCTGCTT-3’) listed on the Switchgear Genomics website (http://switchgeargenomics.com/resources/vector-maps).

  2. Hypoxia reporter assay (LightSwitch Dual Assay System Manual)
    Day 1 (Carry out under a Laminar flow hood)
    1. Coat two white 96-well plates with 1x Poly-D-lysine (50 μl/well) for 2 h at room temperature.
      1. Plate enough wells per plate to carry out in triplicate.
      2. Example samples:
        1. Empty vector (pLightSwitch_Prom) + pTK-Cluc
        2. Wild-type Bmx promoter (pLightSwitch_Prom_BMX) + pTK-Cluc
        3. HRE mutated BMX promoter (pLightSwitch_Prom_BMXmHRE) + pTK-Cluc 
      3. Use one plate per condition (i.e., 2 plates for comparison normoxia to hypoxia).
    2. Wash the plates once with sterile H2O (200 μl/well); plate 5 x 103 HEK293 cells per well in 100 μl of growth media and cultured overnight at 37 °C in 5% CO2.

    Day 2
    Preparation of constructs and reagents
    1. Allow all reagents and plasmids to equilibrate to room temperature.
      Note: Plasmids should be purified using a kit or protocol that will remove endotoxin, such as PureYeild Plasmid System from Promega.
    2. Pre-warm Opti-MEM to 37 °C.

    Transfection (Carry out under a Laminar flow hood)
    1. Carry out transfections according to protocol details (LightSwitch Dual Assay System; link to protocol found above) for a 96-well plate with minimal changes.
      Cypridina TK control construct, pTK-Cluc: 1 ng (One nanogram is used instead of 10 ng due to luciferase saturation)
      pLightSwitch_Prom, pLightSwitch_Prom_BMX or pLightSwitch_Prom_BMXmHRE: 30 ng
      The ratio of FuGENE® HD to plasmid: 3:1 (0.3 μl FuGENE:100 ng DNA per well)
    2. Briefly, the transfection protocol is:
      1. Combine reagents as indicated in the following table and mix well.

      2. Let sit at RT for 30 min.
      3. Gently drop onto seeded wells.
      4. Gently shake.
      5. Plates should be incubated at 37 °C for 24 h in normoxia (5% CO2).

    Day 3 (Under a Laminar flow hood)
    1. Remove the transfection reagents by changing out the media on both plates.
    2. Transfer one plate into hypoxia (5% CO2, 94% N2, and 1% O2) at 37 °C and maintain another one in normoxia.
    3. Incubate both plates for an additional 24 h.
      Note: Incubation time can be varied depending on the induction rate of your favorite gene due to hypoxia.

    Day 4
    Follow the manufacturer's instructions (LightSwitch Dual Assay System) for measuring Renilla (pLightSwitch_Prom vectors) and Cypridina (pTK-Cluc) luciferase activity.
    1. No alterations were made to the protocol detailed by SwitchGear Genomics. Plates were always measured fresh and never frozen.
    2. For hypoxia conditions (all done within the hypoxia chamber):
      1. Adding reagents and incubations are all done within the hypoxia chamber.
      2. Remove the plates from hypoxia and immediately read the luminescence on a plate reader.
    3. For normoxia conditions: Adding reagents and incubation are all done at room temperature on the bench top.

Data analysis

  1. Renilla (pLightSwitch_Prom) luciferase activity is normalized to Cypridina (pTK-Cluc) luciferase activity and adjusted for background luciferase for each condition.
  2. Data can then be graphed as relative luciferase activity or as fold change.(see Figure 1)
    Note: If fold change is determined, the comparison should be to the wild-type promoter under normoxia conditions.


    Figure 1. Example data

Notes

While there may be variation in transfection efficiency from experiment to experiment, the LightSwitch Dual Assay system is designed to improve experimental accuracy by reducing extraneous influences. Thus your reproducibility and variability should be minimal after the Renilla luciferase activity has been normalized to the Cypridina luciferase activity.

Recipes

  1. Terrific Broth
    47.6 g Terrific Broth powder
    4 ml glycerol
    1 L H2O
    Autoclaved at 121 °C for 15 min
  2. 5x Poly-D-lysine Solution
    Dissolve 5 mg Poly-D-lysine in 1 L ddH2O
  3. 1x Poly-D-lysine Solution (Sterile)
    Dilute 200 ml 5x Poly-D-Lysine solution w with 800 ml ddH2O and filter sterilize
  4. Growth media
    DMEM
    10% FBS

Acknowledgments

We thank Navjotsingh Pabla for assistance with protocol development and helpful comments during manuscript preparation. This study was supported by the American Lebanese Syrian Associated Charities, NIH Cancer Center Support Grant P30 CA021765, and R01 CA138744 (to SDB). This study was also supported by funds from the Ohio State University Comprehensive Cancer Center, Pelotonia foundation, and NIH Cancer Center Support Grant P30 CA016058. Authors declare that there are no conflicts of interest or competing interests.

References

  1. Chen, S., Jiang, X., Gewinner, C. A., Asara, J. M., Simon, N. I., Cai, C., Cantley, L. C. and Balk, S. P. (2013). Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases. Sci Signal 6(277): ra40. 
  2. Davis, M. I., Hunt, J. P., Herrgard, S., Ciceri, P., Wodicka, L. M., Pallares, G., Hocker, M., Treiber, D. K. and Zarrinkar, P. P. (2011). Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 29(11): 1046-1051.
  3. He, Y., Luo, Y., Tang, S., Rajantie, I., Salven, P., Heil, M., Zhang, R., Luo, D., Li, X., Chi, H., Yu, J., Carmeliet, P., Schaper, W., Sinusas, A. J., Sessa, W. C., Alitalo, K. and Min, W. (2006). Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis. J Clin Invest 116(9): 2344-2355.
  4. van Oosterwijk, J. G., Buelow, D. R., Drenberg, C. D., Vasilyeva, A., Li, L., Shi, L., Wang, Y. D., Finkelstein, D., Shurtleff, S. A., Janke, L. J., Pounds, S., Rubnitz, J. E., Inaba, H., Pabla, N. and Baker, S. D. (2018). Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia. J Clin Invest 128(1): 369-380. 

简介

缺氧是细胞环境中氧供应减少的一种情况。 细胞氧水平的变化可导致氧调节基因的转录变化。 报告分析用于研究响应环境变化的基因表达改变和修饰。 双报告分析允许同时测量单个细胞内的两个不同基因,从而提高实验准确性。 在该协议中,我们描述了LightSwitch双重测定系统的使用,以测量响应缺氧条件的BMX表达。

【背景】在我们最近的出版物(van Oosterwijk et al。,2018)中,我们试图检测BMX(一种非受体酪氨酸激酶)对索拉非尼治疗的反应。 BMX是一种Tec激酶家族成员,已知与酪氨酸磷酸化蛋白结合并通过与磷脂酰肌醇3,4,5-三磷酸结合而介导膜靶向(PIP3; Chen 等人,2013)。我们发现索拉非尼在有或没有基质细胞支持的急性髓性白血病(AML)细胞系中的直接治疗对BMX的上调没有贡献。以前的研究表明缺血可诱导BMX表达,索拉非尼具有抗血管生成活性(He et al。,2006; Davis et al。,2011)。因此,我们假设索拉非尼的抗血管生成活性导致骨髓内的缺氧环境,从而导致AML中缺氧依赖性BMX上调。在缺氧条件下,我们能够显示许多不同细胞系中BMX表达的显着增加。 BMX启动子的进一步分析在-5005处鉴定出推定的缺氧反应元件(HRE; 5'-ACGTG-3')。为了测试该HRE是否参与缺氧诱导的BMX启动子激活,我们开发了缺氧元件报道分子测定法。报告分析广泛用于整个科学界,以研究响应环境变化的基因表达的改变和修饰。一种获得认可的报告基因检测是双报告基因检测。这种类型的报告分析允许在单个细胞内同时表达和测量两种不同的报道分子,并且已被广泛证明通过减少外来影响来提高实验准确性。其中一种双重检测系统是 LightSwitch双重检测系统。该系统利用RenSP荧光素酶报告基因,一种由SwitchGear Genomics和Cypridina荧光素酶报告基因开发的新型荧光素酶。 RenSP和Cypridina使用不同的底物,从而消除蛋白质与其底物之间的交叉反应。 RenSP与您最喜欢的基因一起用作报告信号,Cypridina与组成型活性启动子一起用作对照信号。

在这里,我们使用LightSwitch双重检测系统来评估BMX HRE参与其低氧条件下的上调。

关键字:荧光素酶, 报告系统检测技术, 低氧, 调控, 转录

材料和试剂

  1. 移液器吸头
  2. 白色96孔板(CELLSTAR ®组织培养板,Greiner Bio One International,目录号:655083)
  3. HEK293(293 [HEK-293]; ATCC,目录号:CRL-1573)
  4. pLightswitch_Prom_BMX(BMX启动子构建; SwitchGear Genomics,目录号:S701154)
  5. pLightswitch_Prom控制向量(SwitchGear Genomics,目录号:S790005)
  6. pTK-Cluc(Cypridina TK control construct; SwitchGear Genomics,目录号:SN0322S)
  7. FuGENE ® HD(Promega,目录号:E2311)
  8. DMEM,高葡萄糖,丙酮酸(Thermo Fisher Scientific,Gibco TM ,目录号:11995065)
  9. QuikChange XL定点诱变试剂盒(安捷伦科技,目录号:200516)
  10. 极好的肉汤粉(BD,Difco TM ,目录号:243820)
  11. 甘油(Sigma-Aldrich,目录号:G9012)
  12. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P6407-5MG)
  13. Opti-MEM(Thermo Fisher Scientific,Gibco TM ,目录号:31985070)
  14. 胎牛血清(Thermo Fisher Scientific,Gibco TM ,目录号:10437028)
  15. LightSwitch双分析系统(SwitchGear Genomics,目录号:DA010)
  16. Poly-D-Lysine hydrobromide(MP Biomedicals,目录号:02150175)
  17. 极好的肉汤(见食谱)
  18. 5x聚-D-赖氨酸溶液(见食谱)
  19. 1x聚-D-赖氨酸溶液(无菌)(见食谱)
  20. 增长媒体(见食谱)

设备

  1. 移液器
  2. 热循环仪(Eppendorf,型号:Mastercycler ® pro S,目录号:950030020)
  3. 台式离心机(Eppendorf,型号:5810 R,目录号:022625101)
  4. 摆动斗转子(Eppendorf,型号:S-4-104,目录号:5820755008)
  5. 高性能离心机(Thermo Fisher Scientific,型号:Sorvall TM RC 6 Plus,目录号:36-101-0816)
  6. 层流罩(赛默飞世尔科技,型号:1300系列II级,A2型,目录号:1323TS)
  7. 37°C,5%CO 2 水套式培养箱(Thermo Fisher Scientific,型号:Heracell VIOS 160i,目录号:51030285)
  8. 37°C非加湿培养箱(Labnet International,型号:311DS,目录号:I5311-DS)
  9. 缺氧室(Coy Lab,型号:O 2 Control InVitro Glove Box,无升级)
    1. 加湿孵化盒
    2. 温度控制
    3. 以0.1%的增量控制O 2 和CO 2
  10. 平板发光计(Molecular Devices,型号:SpectraMax i3x,无升级版)
    标准功能包括:
    1. 读取模式:吸光度,荧光,发光
    2. 波长范围包括:Abs:230-1,000nm,Fl Ex:250-830nm,Fl EM 270-850nm,Lumi:300-850nm

软件

  1. GraphPad Prism(GraphPad软件,版本6)
  2. QuikChange Primer Design( https://www.genomics.agilent.com/primerDesignProgram.jsp )

程序

  1. pLightswitch_Prom_BMX的定点诱变突变BMX启动子内的推定HRE
    1. 设计引物以使用QuikChange Primer Design网站(参考上文)突变BMX启动子的HRE内的2个碱基对(A CG或TG A→TG)。
      文献中已经描述了CG→TA bp的变化,以有效地破坏HRE。
    2. 每个制造商使用 QuikChangeXL定点诱变试剂盒对pLightSwitch_Prom_BMX进行突变协议。
      1. 延长时间:4.5分钟
      2. 用于NZY + 肉汤的替代Difco TM 极好的肉汤
    3. 通过使用Switchgear Genomics网站上列出的启动子插入测序引物(正向:5'-TCCATCAAAACAAAACGAAACAA-3',反向:5'-AGTCGAGCACGTTCATCTGCTT-3')测序来确认突变质粒( http://switchgeargenomics.com/resources/vector-maps )。

  2. 缺氧报告基因检测( LightSwitch双重检测系统手册)
    第1天(在Laminar流罩下进行)
    1. 在室温下将两个白色96孔板与1x聚-D-赖氨酸(50μl/孔)一起涂覆2小时。
      1. 每板镀足够的孔,一式三份进行。
      2. 示例示例:
        1. 空载体(pLightSwitch_Prom)+ pTK-Cluc
        2. 野生型Bmx启动子(pLightSwitch_Prom_BMX)+ pTK-Cluc
        3. HRE突变BMX启动子(pLightSwitch_Prom_BMXmHRE)+ pTK-Cluc 
      3. 每种条件使用一个平板(即,2个平板用于比较常氧和缺氧)。
    2. 用无菌H 2 O(200μl/孔)洗涤板一次;在100μl生长培养基中每孔培养5×10 3个 3 HEK293细胞,并在37℃,5%CO 2 中培养过夜。

    第2天
    构建体和试剂的制备
    1. 让所有试剂和质粒平衡至室温。
      注意:质粒应使用可去除内毒素的试剂盒或方案进行纯化,例如Promega的PureYeild Plasmid System。
    2. 预热Opti-MEM至37°C。

    转染(在Laminar流罩下进行)
    1. 根据协议细节(LightSwitch Dual Assay System;链接到上面找到的方案)进行96孔板的转染,只需极少的改变。
      Cypridina TK对照构建体,pTK-Cluc:1 ng(由于荧光素酶饱和,使用1纳克代替10 ng)
      pLightSwitch_Prom,pLightSwitch_Prom_BMX或pLightSwitch_Prom_BMXmHRE:30 ng
      FuGENE ® HD与质粒的比例:3:1(0.3μlFuGENE:每孔100 ng DNA)
    2. 简言之,转染方案是:
      1. 如下表所示混合试剂并充分混合。

      2. 我们坐在室温下30分钟。
      3. 轻轻地落在种子井上。
      4. 轻轻摇晃。
      5. 平板应在常氧(5%CO 2 )中于37℃温育24小时。

    第3天(在层流罩下)
    1. 通过更换两个平板上的介质来移除转染试剂。
    2. 在37°C将一个平板转移到缺氧(5%CO 2 ,94%N 2 和1%O 2 )并维持另一个一个在常氧中。
    3. 将两个平板孵育24小时。
      注意:孵化时间可能因缺氧导致您喜欢的基因的诱导率而异。

    第4天
    按照制造商的说明( LightSwitch Dual Assay System )测量Renilla(pLightSwitch_Prom载体)和Cypridina (pTK-Cluc)荧光素酶活性。
    1. SwitchGear Genomics详细描述的协议没有改变。板材总是新鲜测量,从不冷冻。
    2. 对于缺氧条件(均在缺氧室内完成):
      1. 添加试剂和孵育都在缺氧室内完成。
      2. 从缺氧处移除平板并立即在平板读数器上读取发光。
    3. 对于常氧条件:添加试剂和孵育均在室温下在工作台上进行。

数据分析

  1. 将Renilla(pLightSwitch_Prom)荧光素酶活性标准化为Cypridina(pTK-Cluc)荧光素酶活性,并针对每种条件调整背景荧光素酶。
  2. 然后可以将数据绘制为相对荧光素酶活性或倍数变化(见图1)
    注意:如果确定倍数变化,则应在常氧条件下与野生型启动子进行比较。


    图1.示例数据

笔记

虽然从实验到实验的转染效率可能存在差异,但LightSwitch Dual Assay系统旨在通过减少外来影响来提高实验准确性。因此,在将海肾荧光素酶活性标准化为Cypridina荧光素酶活性后,您的重现性和可变性应该是最小的。

食谱

  1. 很棒的肉汤
    47.6克极好的肉汤粉
    4毫升甘油
    1 L H 2 O
    在121°C高压灭菌15分钟
  2. 5x聚-D-赖氨酸溶液
    将5mg聚-D-赖氨酸溶解在1L ddH 2 O中
  3. 1x聚-D-赖氨酸溶液(无菌)
    用800 ml ddH 2 O稀释200 ml 5x聚-D-赖氨酸溶液w并过滤灭菌
  4. 成长媒体
    DMEM
    10%FBS

致谢

我们感谢Navjotsingh Pabla在协稿开发方面的帮助以及稿件准备期间的有益评论。该研究得到了美国黎巴嫩叙利亚联合慈善机构,NIH癌症中心支持补助金P30 CA021765和R01 CA138744(致SDB)的支持。该研究还得到了俄亥俄州立大学综合癌症中心,Pelotonia基金会和NIH癌症中心支持基金P30 CA016058的资助。作者声明没有利益冲突或竞争利益。

参考

  1. Chen,S.,Jiang,X.,Gewinner,C.A.,Asara,J.M.,Simon,N.I。,Cai,C.,Cantley,L.C。和Balk,S.P。(2013)。 酪氨酸激酶BMX磷酸化磷酸酪氨酸引发的基序,介导多种受体酪氨酸激酶的激活。 Sci Signal 6(277):ra40。 
  2. Davis,M。I.,Hunt,J.P.,Herrgard,S.,Ciceri,P.,Wodicka,L.M.,Pallares,G.,Hocker,M.,Treiber,D.K。和Zarrinkar,P.P。(2011)。 激酶抑制剂选择性的综合分析。 Nat Biotechnol 29 (11):1046-1051。
  3. He,Y.,Luo,Y.,Tang,S.,Rajantie,I.,Salven,P.,Heil,M.,Zhang,R.,Luo,D.,Li,X.,Chi,H., Yu,J.,Carmeliet,P.,Schaper,W.,Sinusas,AJ,Sessa,WC,Alitalo,K。和Min,W。(2006)。 Bmx / Etk在缺血介导的动脉发生和血管生成中的关键功能。 J Clin Invest 116(9):2344-2355。
  4. van Oosterwijk,JG,Buelow,DR,Drenberg,CD,Vasilyeva,A.,Li,L.,Shi,L.,Wang,YD,Finkelstein,D.,Shurtleff,SA,Janke,LJ,Pounds,S。, Rubnitz,JE,Inaba,H.,Pabla,N。和Baker,SD(2018)。 缺氧诱导的BMX激酶上调介导急性髓性白血病的治疗抵抗。 J Clin Invest 128(1):369-380。 
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引用:Buelow, D. R. and Baker, S. D. D. (2018). Hypoxia Reporter Element Assay. Bio-protocol 8(15): e2951. DOI: 10.21769/BioProtoc.2951.
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