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Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination
免疫沉淀法识别调节蛋白质泛素化的因子:TRF2在人REST4泛素化中的案例研究   

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Abstract

Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination status of the human REST4 protein in cultured cells, a neural alternative splicing isoform of REST (RE-1 silencing transcription factor), that antagonizes the repressive function of REST on neural differentiation and neuron formation. Using this protocol, we show that the telomere binding protein TRF2 stabilizes the expression of the human REST4 by inhibiting its ubiquitination. This indicates that TRF2 plays a positive role in neural differentiation (Ovando-Roche et al., 2014). This protocol is also useful for the detection of ubiquitination of other proteins of interest.

Keywords: TRF2(TRF2), Immunoprecipitation(免疫共沉淀), REST(休息)

Materials and Reagents

  1. Cells and plasmid
    1. TRF2-HEK293T cells: 293T cells (ATCC, catalog number: CRL-3216 ) that have been stably transfected with TRF2 expressing vector
    2. Control-HEK-293T cells: 293T cells (ATCC, catalog number: CRL-3216) that have been stably transfected with empty control vector
    3. Myc-REST4 expressing vector: generated by modifying the Myc-REST plasmid (Huang et al., 2011) as detailed in Ovando-Roche et al. (2014)
    4. HA-ubiquitin expressing vector: (Addgene, catalog number: 17608 )

  2. Antibodies
    1. Anti-TRF2 antibody (EMD Millipore, catalog number: 05-521 )
    2. Anti-HA antibody (Sigma-Aldrich, catalog number: H6908 )
    3. Anti β-Actin antibody (Sigma-Aldrich, catalog number: A5441 )
    4. C-Myc antibody (Santa Cruz, catalog number: SC-40 )
    5. Goat anti-mouse light chain specific (Jackson ImmunoResearch Laboratories, catalog number: 115-035-174 )
    6. Mouse anti-rabbit light chain specific (Jackson ImmunoResearch Laboratories, catalog number: 211-032-171 )
    7. Normal mouse IgG (Santa Cruz, catalog number: SC-2025 )

  3. Reagents
    1. Dulbeccos modified eagles medium (DMEM) (Sigma-Aldrich, catalog number: D5671 )
    2. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
    3. Calcium phosphate transfection kit (Sigma-Aldrich, catalog number: CAPHOS )
    4. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
    5. Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 )
    6. CaCl2 (VWR International, catalog number: 22317.297 )
    7. Dynabeads protein G (Life Technologies, catalog number: 10003D )
    8. EDTA (Sigma-Aldrich, catalog number: ED-1KG )
    9. EGTA (Sigma-Aldrich, catalog number: E4378 )
    10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    11. HEPES (Sigma-Aldrich, catalog number: H3375 )
    12. KCl (VWR International, catalog number: JT3040-1 )
    13. MG132 (Calbiochem, catalog number: 474791 )
    14. MgCl2 (Sigma-Aldrich, catalog number: 63063 )
    15. NaCl (Sigma-Aldrich, catalog number: s-3014 )
    16. NaOH (VWR International, catalog number: 28244.263 )
    17. Nonidet P40 (VWR International, catalog number: E109-100ML )
    18. Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: 93482 )
    19. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
    20. SDS (Sigma-Aldrich, catalog number: 71725 )
    21. Tris-HCl (Sigma-Aldrich, catalog number: T3253 )
    22. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    23. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
    24. Ubiquitin aldehyde (Boston Biochem, catalog number: U-201 )

  4. Buffers
    1. Antibody binding buffer (see Recipes)
    2. HEPES lysis buffer (see Recipes)
    3. 2x electrophoresis sample buffer (see Recipes)

Equipment

  1. 100 mm cell culture dish (Corning, catalog number: 430167 )
  2. Cell scraper (Corning, catalog number: 3010 )
  3. 1.5 ml Eppendorf tubes (StarLab, catalog number: S1615-5500 )
  4. BD Microlance 3 21G needle (BD, catalog number: 301156 )
  5. 37 °C, 5% CO2 forced-air incubator (Binder, model: C150 )
  6. Microcentrifuge (Hettich, model: 200R )
  7. Thermomixer compact (Eppendorf, model: 5350 )
  8. Rotator (Stuart, model: SB3 )
  9. Dynamag 2 magnet (Life Technologies, catalog number: 12321D )

Procedure

  1. Culture TRF2-HEK293T and control cells in 100 mm dishes with a standard medium (DMEM containing 10% FBS). Upon reaching 60-70% confluency, co-transfect the cells with plasmids Myc-REST4/HA-Ubiquitin using Calcium phosphate transfection kit following manufacturer’s instruction.
  2. 48 h after the transfection, treat the cells with 20 µM MG132 (protect from light) in the standard medium and incubate for 5 h at 37 °C, 5% CO2.
  3. Remove medium and wash cells with PBS, then lyse cells in a 100 mm dish with 600 µl of chilled HEPES lysis buffer (incubate on ice/4 °C for 4 min before scraping to increase protein yield).
  4. Collect cell lysate in an Eppendorf tube and pass lysate through 21G needle ~25 times to aid lysis and shred DNA.
  5. Add ubiquitin aldehyde to the lysate to a final concentration of 1 µM and mix sample on a rotator at 20 rpm for 15 min at 4 °C.
  6. Centrifuge the sample at 12,000 x g for 15 min at 4 °C to pellet cell debris and transfer the supernatant to a new tube.
  7. Pre-clear samples with 50 µl of protein G Dynabeads for 1 h at 4 °C with a rotator at 20 rpm to remove proteins that non-specifically stick to beads.
  8. Bind antibodies to beads for immunoprecipitation (IP):
    1. Vortex Dynabeads stock for >30 sec to re-suspend beads.
    2. Dispense 50 µl of beads per IP reaction.
    3. Place on magnet to separate beads from solution, aspirate out solution.
    4. Dilute 3 µg Myc antibody in 500 µl of binding buffer and do the same for normal mouse IgG control.
      Note: Since Myc antibody was raised from mouse, normal mouse IgG is used as negative control.
    5. Bind antibodies to the beads by incubating 30 min at room temperature and 30 min at 4 °C with shaking.
  9. Quantify the pre-cleared sample for protein concentration and prepare 1 mg protein per IP and 20 µg protein per input.
  10. After antibody-beads binding, place tube containing beads onto magnet and remove binding solution. Wash beads once with binding solution to remove any excess antibody.
  11. Transfer 1 mg of pre-cleared proteins into the beads that have been linked to Myc antibody or normal mouse IgG. Mix samples and incubate for 2 h at 4 °C with rotation at 20 rpm to perform IP.
  12. Following formation of the beads-antibody-antigen complex, place samples back onto magnet to remove the supernatant.
  13. Re-suspend beads in 400 µl lysis buffer and place back onto magnet, remove supernatant as before.
  14. Repeat step 13 two more times to wash beads of any unspecific binding.
  15. After final wash re-suspend beads-antibody-antigen complex in 100 µl lysis buffer and transfer to a clean tube.
  16. Place the samples in magnet and remove the supernatant. Re-suspend complex pellet in 40 µl 2x electrophoresis buffer, vortex harshly and pulse spin in microcentrifuge to collect all the solution to the bottom of the tube.
  17. Heat the above sample at 70 °C for 10 min with shaking in a thermomixer.
  18. Place the sample back onto magnet for a final time and transfer supernatant to a new tube (IP tube).
  19. Boil the IP samples and Input samples (20 µg of lysate directly from step 9 for each sample) at 95 °C for 5 min.
  20. Analyze the samples via Western blotting.
    1. Specificity and quality of IP: In order to verify the function of TRF2 on REST4 ubiquitination, it is necessary to ensure that similar amount of Myc-REST4 proteins are precipitated between TRF2 expressing and control cells and that this precipitation is specific. Therefore, IP samples are tested with anti-c-Myc (1:1,000) antibody as shown in Figure 1.
    2. Effect of TRF2 on REST4 ubiquitination: IP samples are also tested for ubiquitin binding with anti-HA (1:1,000 dilution) antibody as ubiquitin is tagged with HA epitope.
    3. To ensure the correct protein expression and loading, all cell lysates without immunoprecipitation (input samples) are tested with anti-c-Myc (1:1,000), anti-TRF2 (1:500) and anti-β-actin (1:5,000) antibodies.

Representative Data

  1. Here is a representative result showing that ubiquitination of REST4 is clearly detected in the cells that were co-transfected with Myc-REST4 and HA-tagged ubiquitin (HA-Ub) and that expression of TRF2 reduces the ubiquitination of REST4 in these cells. The results suggest that TRF2 stabilizes REST4 expression by inhibition of its ubiquitin-proteasomal degradation (Ovando-Roche et al., 2014).


    Figure 1. TRF2 inhibits human REST4 ubiquitination. Control and TRF2 stably overexpressing HEK293T cells were co-transfected with Myc-REST4 and HA-ubiquitin (HA-Ub) expression vectors. Myc immunoprecipitates (IP) or whole cell lysates (input) were immunobloted with the indicated antibodies. Immunoblot is representative of two independent experiments.

Recipes

  1. Antibody binding buffer
    PBS + 0.01% Tween 20 (1 µl Tween 20 to 10 ml PBS)
  2. HEPES lysis buffer
    20 mM HEPES (pH 7.4)
    10 mM KCl
    100 mM NaCl2
    1 mM MgCl2
    1 mM EDTA
    0.1 mM EGTA
    0.5 mM CaCl2
    Before use add following reagents to the indicated final concentrations: 1x protease inhibitor cocktail, 0.2 mM phenylmethanesulfonyl fluoride, 0.1% Triton X-100 and 0.5% Nonidet P40
  3. 2x electrophoresis sample buffer
    2.5 ml of 0.5 M Tris-HCl (pH 6.8)
    2 ml of glycerol
    4 ml of 10% SDS (w/v)
    0.94 ml dH2O
    0.16 ml 1.25 % bromophenol blue
    Total volume: 9.5 ml
    To prepare 1 ml, add 50 µl of -mercaptoethanol to 950 µl 2x sample buffer

Acknowledgments

We are grateful to Dr. Shideng Bao from Lerner Research Institute for the REST plasmid. The work was supported by the funding of BBSRC, MRC (G0802537) and Genesis Research Trust. This work is adapted from the work previously published (Ovando-Roche et al., 2014).

References

  1. Bhattacharyya, S., Yu, H., Mim, C. and Matouschek, A. (2014). Regulated protein turnover: snapshots of the proteasome in action. Nat Rev Mol Cell Biol 15(2): 122-133.
  2. Huang, Z., Wu, Q., Guryanova, O. A., Cheng, L., Shou, W., Rich, J. N. and Bao, S. (2011). Deubiquitylase HAUSP stabilizes REST and promotes maintenance of neural progenitor cells. Nat Cell Biol. 13:142-152.
  3. Ovando-Roche, P., Yu, J. S., Testori, S., Ho, C. and Cui, W. (2014). TRF2-mediated stabilization of hREST4 is critical for the differentiation and maintenance of neural progenitors. Stem Cells 32(8): 2111-2122.
  4. Ristic, G., Tsou, W. L. and Todi, S. V. (2014). An optimal ubiquitin-proteasome pathway in the nervous system: the role of deubiquitinating enzymes. Front Mol Neurosci 7: 72.
  5. Weissman, A. M., Shabek, N. and Ciechanover, A. (2011). The predator becomes the prey: regulating the ubiquitin system by ubiquitylation and degradation. Nat Rev Mol Cell Biol 12(9): 605-620.

简介

泛素化是泛素 - 蛋白酶体通路的第一步,其调节细胞的内环境稳定功能,并且是酶,蛋白质翻译后修饰过程,其中通过一组三种泛素酶将泛素转移到靶蛋白质底物(Weissman et al。,2011; Bhattacharyya et al。,2014; Ristic 等人,2014)。考虑到这一过程的重要性,似乎可能的是,泛素化受到许多因素的严格控制,并且调节机制是蛋白质特异性的。用于检测特异性蛋白泛素化的测定将使我们能够检查因子是否具有调节该蛋白的泛素化的功能。在这里我们描述一个协议,检测人类REST4蛋白在培养细胞中的神经选择性剪接异构体(RE-1沉默转录因子),拮抗REST对神经分化和神经元形成的镇压功能的泛素化状态。使用这个协议,我们显示端粒结合蛋白TRF2通过抑制其泛素化稳定人类REST4的表达。这表明TRF2在神经分化中发挥阳性作用(Ovando-Roche等人,2014)。该方案还可用于检测其他感兴趣的蛋白质的泛素化。

关键字:TRF2, 免疫共沉淀, 休息

材料和试剂

  1. 细胞和质粒
    1. TRF2-HEK293T细胞:用TRF2表达载体稳定转染的293T细胞(ATCC,目录号:CRL-3216)
    2. 对照-HEK-293T细胞:293T细胞(ATCC,目录号:CRL-3216) 其已经用空的对照载体稳定转染
    3. Myc-REST4表达载体:通过修饰Myc-REST质粒产生   (Huang et al。,2011),如在Ovando-Roche等人(2014)中详述的。
    4. HA-泛素表达载体:(Addgene,目录号:17608)

  2. 抗体
    1. 抗TRF2抗体(EMD Millipore,目录号:05-521)
    2. 抗HA抗体(Sigma-Aldrich,目录号:H6908)
    3. 抗β-肌动蛋白抗体(Sigma-Aldrich,目录号:A5441)
    4. C-Myc抗体(Santa Cruz,目录号:SC-40)
    5. 山羊抗小鼠轻链特异性(Jackson ImmunoResearch Laboratories,目录号:115-035-174)
    6. 小鼠抗兔轻链特异性(Jackson ImmunoResearch Laboratories,目录号:211-032-171)
    7. 正常小鼠IgG(Santa Cruz,目录号:SC-2025)

  3. 试剂
    1. Dulbeccos改良的Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D5671)
    2. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)
    3. 磷酸钙转染试剂盒(Sigma-Aldrich,目录号:CAPHOS)
    4. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
    5. 溴酚蓝(Bio-Rad Laboratories,目录号:161-0404)
    6. CaCl 2 (VWR International,目录号:22317.297)
    7. Dynabeads蛋白G(Life Technologies,目录号:10003D)
    8. EDTA(Sigma-Aldrich,目录号:ED-1KG)
    9. EGTA(Sigma-Aldrich,目录号:E4378)
    10. 甘油(Sigma-Aldrich,目录号:G5516)
    11. HEPES(Sigma-Aldrich,目录号:H3375)
    12. KCl(VWR International,目录号:JT3040-1)
    13. MG132(Calbiochem,目录号:474791)
    14. MgCl <2> (Sigma-Aldrich,目录号:63063)
    15. NaCl(Sigma-Aldrich,目录号:s-3014)
    16. NaOH(VWR International,目录号:28244.263)
    17. Nonidet P40(VWR International,目录号:E109-100ML)
    18. 苯基甲磺酰氟(Sigma-Aldrich,目录号:93482)
    19. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
    20. SDS(Sigma-Aldrich,目录号:71725)
    21. Tris-HCl(Sigma-Aldrich,目录号:T3253)
    22. Triton X-100(Sigma-Aldrich,目录号:T8787)
    23. 吐温20(Sigma-Aldrich,目录号:P9416)
    24. 泛素醛(Boston Biochem,目录号:U-201)

  4. 缓冲区
    1. 抗体结合缓冲液(参见配方)
    2. HEPES裂解缓冲液(见配方)
    3. 2x电泳样品缓冲液(见配方)

设备

  1. 100mm细胞培养皿(Corning,目录号:430167)
  2. 细胞刮刀(Corning,目录号:3010)
  3. 1.5ml Eppendorf管(StarLab,目录号:S1615-5500)
  4. BD Microlance 3 21G针(BD,目录号:301156)
  5. 37℃,5%CO 2强制空气培养箱(Binder,型号:C150)中。
  6. 微量离心机(Hettich,型号:200R)
  7. 热固性混合机(Eppendorf,型号:5350)
  8. 旋转器(Stuart,型号:SB3)
  9. Dynamag 2磁体(Life Technologies,目录号:12321D)

程序

  1. 培养物TRF2-HEK293T和对照细胞在具有标准培养基(含有10%FBS的DMEM)的100mm皿中。 达到60-70%融合后,使用磷酸钙转染试剂盒按照制造商的说明书用质粒Myc-REST4/HA-泛素共转染细胞。
  2. 转染后48小时,用标准培养基中的20μMMG132(防止光照)处理细胞,并在37℃,5%CO 2下孵育5小时。
  3. 除去培养基并用PBS洗涤细胞,然后在600毫升冷冻的HEPES裂解缓冲液(在冰上/4℃孵育4分钟,然后刮,以提高蛋白质产量)在100毫米的培养皿中溶解细胞。
  4. 收集细胞裂解液在Eppendorf管中,并通过21G针的裂解液〜25次,以帮助裂解和切碎DNA。
  5. 向裂解物中加入泛素醛至终浓度为1μM,并在旋转器上以20rpm在4℃下混合样品15分钟。
  6. 在4℃下将样品以12,000xg离心15分钟以沉淀细胞碎片,并将上清液转移到新管中。
  7. 使用50μl蛋白G Dynabeads在4℃下用旋转器以20rpm预清洁样品1小时以除去非特异性粘附于珠粒的蛋白质。
  8. 将抗体结合到珠上用于免疫沉淀(IP):

    1. 每次IP反应分配50μl珠子
    2. 放在磁铁上以从溶液中分离珠子,吸出溶液
    3. 在500μl结合缓冲液中稀释3μgMyc抗体,并对正常小鼠IgG对照进行相同操作。
      注意:由于Myc抗体来自小鼠,正常小鼠IgG用作阴性对照。
    4. 通过在室温下孵育30分钟和在4℃下振荡孵育30分钟,将抗体结合至珠。
  9. 量化预先清除的样品的蛋白质浓度,并准备1毫克蛋白每IP和每个输入20微克蛋白质
  10. 在抗体珠结合后,将含有珠的管置于磁体上并除去结合溶液。 用结合溶液洗涤珠一次,以除去任何过量的抗体
  11. 将1mg预先纯化的蛋白质转移到已经连接Myc抗体或正常小鼠IgG的珠中。 混合样品,并在4℃下以20rpm旋转孵育2小时以进行IP。
  12. 在形成珠 - 抗体 - 抗原复合物后,将样品放回到磁体上以除去上清液。
  13. 重悬在400μl裂解缓冲液中的珠子,并放回磁铁,如上所述除去上清液。
  14. 重复步骤13两次以洗涤任何非特异性结合的珠子
  15. 最终洗涤后,将珠子 - 抗体 - 抗原复合物在100μl裂解缓冲液中重悬,并转移到干净的管中
  16. 将样品放在磁铁中,取出上清液。 重悬在40μl2X电泳缓冲液中的复杂沉淀,剧烈涡旋和在微量离心机中的脉冲旋转收集所有的溶液到管的底部。
  17. 将上述样品在70℃下在热混合器中摇动加热10分钟
  18. 将样品放回磁体最后一次,并将上清液转移到新管(IP管)
  19. 在95℃下煮沸IP样品和输入样品(每个样品直接从步骤9得到20μg裂解物)5分钟。
  20. 通过蛋白质印迹分析样品。
    1. IP的具体性和质量:为了验证的功能 TRF2对REST4的泛素化,有必要保证类似 在表达TRF2之间沉淀Myc-REST4蛋白的量 和对照细胞,并且该沉淀是特异性的。 因此,IP   用抗c-Myc(1:1,000)抗体测试样品,如图所示 图1.
    2. TRF2对REST4泛素化的影响:IP样品是 还测试了与抗HA的泛素结合(1:1000稀释) 抗体作为泛素用HA表位标记。
    3. 保证 正确的蛋白表达和加载,所有细胞裂解物无 用抗c-Myc测试免疫沉淀(输入样品) (1:1,000),抗-TRF2(1:500)和抗β-肌动蛋白(1:5,000)抗体。

代表数据

  1. 这是一个代表性的结果,显示在与Myc-REST4和HA标记的泛素(HA-Ub)共转染的细胞中清楚地检测到REST4的泛素化,并且TRF2的表达减少了这些细胞中REST4的泛素化。结果表明TRF2通过抑制其泛素 - 蛋白酶体降解来稳定REST4表达(Ovando-Roche等人,2014)。


    图1.TRF2抑制人REST4泛素化。将对照和TRF2稳定过表达的HEK293T细胞与Myc-REST4和HA-泛素(HA-Ub)表达载体共转染。将Myc免疫沉淀(IP)或全细胞裂解物(输入)用所示抗体免疫印迹。免疫印迹是两次独立实验的代表。

食谱

  1. 抗体结合缓冲液
    PBS + 0.01%吐温20(1μl吐温20至10ml PBS)
  2. HEPES裂解缓冲液
    20mM HEPES(pH7.4) 10 mM KCl
    100mM NaCl sub 2
    1mM MgCl 2
    1mM EDTA
    0.1 mM EGTA
    0.5mM CaCl 2·h/v 在使用前,加入下列试剂至指定的最终浓度:1×蛋白酶抑制剂混合物,0.2mM苯基甲磺酰氟,0.1%Triton X-100和0.5%Nonidet P40
  3. 2x电泳样品缓冲液
    2.5ml 0.5M Tris-HCl(pH 6.8)
    2ml甘油 4ml 10%SDS(w/v) 0.94ml dH 2 O 2 / 0.16ml 1.25%溴酚蓝
    总体积:9.5 ml
    准备1毫升,加入50微升巯基乙醇到950微升2×样品缓冲液

致谢

我们感谢Lerner研究所的Shideng Bao博士的REST质粒。 这项工作得到了BBSRC,MRC(G0802537)和Genesis研究信托基金的资助。 这项工作改编自以前发表的作品(Ovando-Roche等人,2014年)。

参考文献

  1. Bhattacharyya,S.,Yu,H.,Mim,C.and Matouschek,A。(2014)。 受调节的蛋白质周转:蛋白酶体活动的快照。 Nat Rev Mol Cell Biol 15(2):122-133
  2. Huang,Z.,Wu,Q.,Guryanova,O.A。,Cheng,L.,Shou,W.,Rich,J.N。和Bao,S。 Deubiquitylase HAUSP稳定REST并促进维护 的神经祖细胞。 Nat Cell Biol。 13:142-152
  3. Ovando-Roche,P.,Yu,J.S.,Testori,S.,Ho,C.and Cui,W.(2014)。 TRF2介导的hREST4的稳定化对于神经祖细胞的分化和维持至关重要。 干细胞 32(8):2111-2122。
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引用:Cui, W. and Ovando-Roche, P. (2015). Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination. Bio-protocol 5(13): e1525. DOI: 10.21769/BioProtoc.1525.
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