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Assessment of TCR-induced Sumoylation of PKC-θ
评估TCR诱导的PKC-θ类泛素化(Sumoylation)修饰    

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Abstract

Sumoylation controls many cellular processes. Protein kinase C-θ (PKC-θ), a member of the Ca2+-independent PKC subfamily of kinases, serves as a regulator of T cell activation by mediating the T cell antigen receptor (TCR)- and coreceptor CD28-induced activation of the transcription factors NF-κB and AP-1 and, to a lesser extent, NFAT, and, subsequently, interleukin 2 (IL-2) production and T cell proliferation. We recently proved that TCR-induced sumoylation of PKC-θ is required for its function in T cells (Wang et al., 2015). Here we describe the method to analyze TCR-induced sumoylation of overexpressed or endogenous PKC-θ, which is carried out by immunoprecipitation of PKC-θ followed by immunoblotting with anti-SUMO1 antibody.

Keywords: TCR(TCR), Sumoylation(SUMO化), PKC-θ(PKC), T cell activation(T细胞的活化)

Background

Like ubiquitination, sumoylation is the process of covalently modifying a target protein with SUMO. To disrupt the non-covalent interactions and to detect sumoylation specifically on the protein of interest, a stringent condition for cell lysis, immunoprecipitation and washing should be used. However, lysing cells with lysis buffer containing 1% or more SDS yields highly viscous cell lysates, making it difficult to proceed to the immunoprecipitation and immunoblotting steps. Here we describe an alternative lysing-denaturing procedure. Firstly, cells were lysed in lysis buffer supplemented with the SUMO specific proteases inhibitor N-ethylmaleimide. After centrifugation, 1% SDS was added into the supernatant of the cell lysates. The lysates were diluted 10-folds with lysis buffer supplemented with N-ethylmaleimide and subjected to immunoprecipitation. This protocol could also be adopted to detect other ubiquitin-like modification such as neddylation.

Materials and Reagents

  1. Corning 50 ml conical tube (Corning, catalog number: 430829 )
  2. 1.5 ml microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
  3. MACS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  4. MACS separators (Miltenyi Biotec, catalog number: 130-042-501 )
  5. Raji B cells (ATCC, catalog number: CCL86 )
  6. Jurkat T cells, Clone E6.1 (ATCC, catalog number: TIB-152 )
  7. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175095 )
  8. Ficoll (Tbdscience, catalog number: HY2015 )
  9. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  10. Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sangon Biotech, catalog number: A610185 )
  11. Bovine serum albumin (BSA)
  12. CD4 microbeads (Miltenyi Biotec, catalog number: 130-045-101 )
  13. Superantigen SEE (Toxin Technology, catalog number: ET404 )
  14. RPMI-1640 medium (GE Healthcare, HyCloneTM, catalog number: SH30027.01 )
  15. Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  16. Antibiotic-antimycotic solution 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  17. N-ethylmaleimide (Sigma-Aldrich, catalog number: E1271-5G )
  18. Sodium dodecyl sulfate (SDS) (Sangon Biotech, catalog number: A600485 )
  19. Antibodies
    Anti-CD3 (affymetrix, eBioscience, catalog number: 16-0037-85 )
    Anti-CD28 (affymetrixeBioscience, catalog number: 16-0289-85 )
    Goat anti-mouse IgG (Jackson ImmunoResearch, catalog number: 115-001-003 )
    Anti-Flag (Sigma-Aldrich, catalog number: F3165 )
    Anti-PKC-θ (Santa Cruz Biotechnology, catalog number: sc-1875 ; BD, Transduction LaboratoriesTM, catalog number: 610089 )
    Anti-SUMO1 (Santa Cruz Biotechnology, catalog number: sc-9060 )
  20. Tris (Sangon Biotech, catalog number: A600194 )
  21. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  22. Nonidet P40 (Sangon Biotech, catalog number: A600385 )
  23. Aprotinin (EMD Millipore, catalog number: 616370 )
  24. Leupeptin (Calbiochem, catalog number: 108976 )
  25. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  26. Sodium pyrophosphate tetrabasic decahydrate (NaPPi) (Sigma-Aldrich, catalog number: S6422 )
  27. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: 567540 )
  28. Protein G sepharose (GE Healthcare, catalog number: 17-0618-01 )
  29. Lysis buffer (see Recipes)

Equipment

  1. Haemocytometer (Beckman Coulter, model: Z1 COULTER COUNTER )
  2. Humidified CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM 3111 )
  3. Laminar air flow bio-safety cabinet (ESCO Micro, model: Airstream class II )
  4. Centrifuge (Eppendorf, model: 5417R )

Software

  1. ImageJ software

Procedure

  1. Cell preparation and cell culture
    1. Dilute 500 ml blood with 500 ml HBSS, carefully layer 35 ml of diluted blood over 15 ml of Ficoll in a 50 ml conical tube.
    2. Centrifuge at 350 x g for 40 min at 25 °C in a swinging-bucket rotor without brake.
    3. Aspirate the upper layer leaving the mononuclear cell layer undisturbed.
    4. Carefully transfer the mononuclear cell layer to a new 50 ml conical tube. Fill the tube with HBSS, mix, and centrifuge at 300 x g for 10 min at 25 °C.
    5. Carefully remove supernatant. Resuspend the cell pellet in 50 ml HBSS and centrifuge at 200 x g for 15 min at 25 °C.
    6. Carefully remove supernatant. Resuspend cell pellet in 10 ml PBS containing 2 mM EDTA and 0.5% BSA. Determine cell number.
    7. Centrifuge cell suspension at 300 x g for 10 min at 25 °C. Aspirate supernatant completely.
    8. Resuspend cell pellet in 80 µl of PBS containing 2 mM EDTA and 0.5% BSA per 107 cells, add 20 µl of CD4 microbeads per 107 cells. Mix and incubate for 15 min in the refrigerator at 4 °C.
    9. Wash cells by adding 2 ml of PBS containing 2 mM EDTA and 0.5% BSA per 107 cells and centrifuge at 300 x g for 10 min at 25 °C.
    10. Remove the supernatant and resuspend up to 108 cells in 500 µl of PBS containing 2 mM EDTA and 0.5% BSA. Then do the magnetic separation according to the CD4 microbeads data sheet to enrich the CD4+ T cells, and save the flow through CD4- fraction of the mononuclear cells as primary antigen-presenting cells (APCs), which is contaminated with very low percentage of CD8+ T cells but not affecting the following stimulation.
    11. Culture the CD4+ T cells or Jurkat T cells, primary APCs or Raji B cells, in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solution.
  2. Cell stimulation
    1. Superantigen (SEE) stimulation
      1. Incubate primary APCs (1 x 107) or Raji B cells (1 x 107) with or without 500 ng/ml (primary APCs) or 100 ng/ml (Raji B) SEE in 200 μl final volume of RPMI-1640 medium (10% FBS) in a 1.5 ml microcentrifuge tube for 30 min at 37 °C. Then centrifuge the cells at 300 x g for 3 min at 25 °C and wash the pellet with ice cold PBS to obtain SEE-loaded APC-SEE or Raji B-SEE cells.
      2. Wash primary CD4+ or Jurkat T cells (1 x 107) with 1 ml ice cold PBS once and resuspend in 200 μl final volume of RPMI-1640 medium, and incubate on ice for 15 min.
      3. Mix APC-SEE or Raji-B-SEE with T cells at a 1 to 1 ratio (in 400 µl final volume), spin down at 4 °C, 300 x g, 1 min. Keep the cell pellet in the medium and immediately incubate the cell pellet for 1, 5, 15, 30 min at 37 °C.
    2. Antibody stimulation
      1. Resuspend Jurkat T (1 x 107) or human primary CD4+ cells (1 x 107) in 200 μl of serum free RPMI. Incubate on ice for 10 min. Then add 10 μg/ml anti-CD3 and/or 2 μg/ml anti-CD28 mAbs, place the mixture on ice for 5 min, followed by crosslinking with goat-anti-mouse IgG (10 μg/ml) and immediately incubate the cells at 37 °C for 1, 5, 15, 30 min .
  3. Centrifuge the cells at 350 x g for 3 min, carefully remove the supernatant and lyse stimulated cells in 120 μl lysis buffer (Xie et al., 2013) containing 20 mM N-ethylmaleimide (NEM) on ice for 10 min.
  4. Clear the lysate by centrifugation (8,500 x g, 10 min, 4 °C), then add 1% SDS (v/v) to the supernatants and dissociate the immunoprecipitated proteins by heating at 90 °C for 10 min.
  5. Dilute lysate obtained in step 4 ten-folds with lysis buffer, and immunoprecipitate Flag-tagged PKC-θ or endogenous PKC-θ with anti-Flag M2 or goat-anti-PKC-θ Abs, respectively, at 4 °C overnight with rotation. Anti-IgG immunoprecipitation and IPs from lysates of unstimulated cells are done in parallel as negative controls. Then add 30 µl protein G beads (50% [v/v] in PBS) and incubate for 2-4 h at 4 °C with rotation.
  6. Centrifuge at 8,000 x g for 5 min at 4 °C, remove the supernatant carefully and wash the immunoprecipitates extensively by adding 1 ml lysis buffer containing 20 mM NEM and vortexing for 3-5 min.
  7. Repeat the wash step for another 4 times. Then subject the samples to SDS-PAGE, and immunoblot with anti-SUMO1 (1:1,000) or mouse-anti-PKC-θ (1:1,000) Abs. A representative immunoblotting is shown in the Representative data section (Figure 5).

Data analysis

  1. To exclude the non-specific bands detected by the anti-SUMO1 antibody, a set of negative controls including anti-IgG immunoprecipitates (IPs) and IPs from lysates of unstimulated cells should be included in the pilot experiment. The system works if the signal of the control lanes is barely detectable and the cell stimulation is successful.
    1. Analyze signals with ImageJ software as followed:
      Click ‘File’ and ‘Open’, then choose the image.
    2. Click the ‘Rectangular’ tool, a box with yellow lines appears. Drag around the signals to define the area to be analyzed. Click ‘Ctrl+1’ to select the first line (Figure 1).


      Figure 1. Selection of the sumoylated bands of PKC-θ in ImageJ

    3. Move the box with arrow keys to next lane without changing the size of the box, click ‘Ctrl+2’ to select next lane. Repeat this step till all lanes are selected.
    4. Move the box with arrow keys to a blank area, click ‘Ctrl+2’ to select the background.
    5. Click ‘Ctrl+3’ to plot lanes. A series of plots will appear in a new window (Figure 2).


      Figure 2. The window of ImageJ after Clicking ‘Ctrl+3’

    6. Select ‘Wand tool’ and click the area of each plot (Figure 3). A new window opens with the volume data representing the pixel intensity inside a defined boundary. Subtract background pixel intensity (using value of blank area) from all values to obtain the relative intensity values (Figure 4).


      Figure 3. Selection of the ‘Wand tool’


      Figure 4. The interface of ImageJ after analysis

    7. Quantify signals of PKC-θ blot in the similar way.
    8. Normalize the SUMO1 value of each lane with its corresponding PKC-θ value.

Representative data



Figure 5. In vivo assay of the sumoylation of PKC-θ (PKC-θ-SUMO1) in human primary CD4+ T cells. Freshly isolated human primary CD4+ T cells were stimulated for 0-30 min (above lanes) with SEE-pulsed human APCs (APC + SEE), at a ratio of 1:1, assessed by immunoblot analysis (IB) of proteins immunoprecipitated (IP) with anti-PKC-θ (top) and of whole-cell lysates (WCL) without immunoprecipitation (bottom), probed with antibodies to various molecules (left margin); actin serves as a loading control (Wang et al., 2015).

Notes

  1. Larger apparent molecular size of the protein should be anticipated due to multiple SUMO attachment sites. Thus a gel with a lower concentration of acrylamide (i.e., 8%) is recommended.
  2. It is important that cells should be lysed as quickly as possible after stimulation, since sumoylation often occurs transiently and is easily deconjugated by the SUMO-specific proteases. Moreover, 20 mM of N-ethylmaleimide, a chemical inhibitor of these proteases is recommended to be supplemented in the lysis buffer in both the lysing and washing steps during the immunoprecipitation procedure.
  3. Since the signal of the sumoylated bands of endogenous PKC-θ could be very weak, overnight incubation of the membrane with anti-SUMO1 Ab at 4 °C and longer exposure time when visualizing the immunoblot signals are highly recommended.

Recipes

  1. Lysis buffer
    20 mM Tris–HCl (pH 7.5)
    150 mM NaCl
    5 mM EDTA
    1% Nonidet P40
    10 μg/ml aprotinin
    10 μg/ml leupeptin
    1 mM PMSF
    5 mM NaPPi
    1 mM Na3VO4

Acknowledgments

This protocol is modified from the in vivo ubiquitination assay (Wertz et al., 2004). Supported by the National Natural Science Foundation of China (31170846) and the Ministry of Science and Technology of China (2013CB835300).

References

  1. Wang, X. D., Gong, Y., Chen, Z. L., Gong, B. N., Xie, J. J., Zhong, C. Q., Wang, Q. L., Diao, L. H., Xu, A., Han, J., Altman, A. and Li, Y. (2015). TCR-induced sumoylation of the kinase PKC-θ controls T cell synapse organization and T cell activation. Nat Immunol 16(11): 1195-1203.
  2. Wertz, I. E., O'Rourke, K. M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., Wu, P., Wiesmann, C., Baker, R., Boone, D. L., Ma, A., Koonin, E. V. and Dixit, V. M. (2004). De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430(7000): 694-699.
  3. Xie, J. J., Liang, J. Q., Diao, L. H., Altman, A. and Li, Y. (2013). TNFR-associated factor 6 regulates TCR signaling via interaction with and modification of LAT adapter. J Immunol 190(8): 4027-4036.

简介

植物细胞壁主要由多糖纤维素,半纤维素和果胶组成。这些组分的结构和组成复杂性对于确定植物生长期间的细胞壁功能是重要的。此外,细胞壁结构限定了植物来源的生物质的多种功能性质,例如食品的流变性质和用于生产纤维素生物燃料的原料适用性。分子生物学实验室中细胞壁化学的典型表征包括用于定量半纤维素和果胶衍生单体的温和酸水解和通过Updegraff方法对纤维素的单独分析。我们采用了一个简化的"一步两步"水解方案,允许通过配对的脉冲安培检测(HPAEC-PAD)的高效阴离子交换层析同时测定纤维素含量,中性糖和糖醛酸样品。在我们的工作中,该方案已经在很大程度上替代了Updegraff纤维素定量和用2μMTFA水解以在微量级上测定基质多糖组成。

[背景] 是基于在121℃下在4%(w/v)硫酸中水解的样品的配对分析。一组样品首先用72%(w/w)硫酸预处理以使纤维素膨胀并使其易于稀释酸水解(图1中的Saeman水解; Saeman等人,1945)。另一组样品不进行这种预处理,导致非结晶基质多糖的水解(图1中的基质水解)。从每个水解制度中回收的葡萄糖的比较允许纤维素量的计算,其与更劳动密集的Updegraff(1969)方案(Bauer和Ibáñez,2014)很好地一致。除了葡萄糖(Glc),源自基质多糖的其它糖可以从基质水解样品中量化(Gao等人,2014)。因此,使用相对少的手动操作,可以从两个水解样品和总共四个HPAEC-PAD实验定量基质单糖和纤维素。尽管该方案需要进行色谱分离,但准备样品所需的"实际操作"时间大大减少使得该技术非常适合于高通量分析。虽然我们已经发现,对于鼠李糖(Rha),阿拉伯糖(Ara),甘露糖(Man)和木糖(Xyl)的稳定和可重现的分析需要多个HPAEC-PAD运行,报道描述同时定量单一的中性和酸性糖运行可以允许进一步改进该协议的吞吐量(Zhang等人,2012; Voiniciuc和Grünl,2016)。此处描述的协议步骤如图1所示。



图1.协议概述。 AIR =酒精不溶残渣。...

关键字:TCR, SUMO化, PKC, T细胞的活化

材料和试剂

  1. 康宁50ml锥形管(Corning,目录号:430829)
  2. 1.5ml微量离心管(Corning,Axygen ,目录号:MCT-150-C)
  3. MACS柱(Miltenyi Biotec,目录号:130-042-401)
  4. MACS分离器(Miltenyi Biotec,目录号:130-042-501)
  5. Raji B细胞(ATCC,目录号:CCL86)
  6. Jurkat T细胞,克隆E6.1(ATCC,目录号:TIB-152)
  7. Hanks平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM ,目录号:14175095)
  8. Ficoll(Tbdscience,目录号:HY2015)
  9. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  10. 乙二胺四乙酸二钠盐(EDTA)(Sangon Biotech,目录号:A610185)
  11. 牛血清白蛋白(BSA)
  12. CD4微珠(Miltenyi Biotec,目录号:130-045-101)
  13. 超抗原SEE(Toxin Technology,目录号:ET404)
  14. RPMI-1640培养基(GE Healthcare,HyClone ,目录号:SH30027.01)
  15. 热灭活的胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  16. 抗生素 - 抗真菌溶液100x(Thermo Fisher Scientific,Gibco TM ,目录号:15240062)
  17. N' - 乙基马来酰亚胺(Sigma-Aldrich,目录号:E1271-5G)
  18. 十二烷基硫酸钠(SDS)(Sangon Biotech,目录号:A600485)
  19. 抗体
    抗CD3( affymetris, eBioscience,目录号:16-0037-85) /> 抗CD28( affymetris ,eBioscience,目录号:16-0289-85) /> 山羊抗小鼠IgG(Jackson ImmunoResearch,目录号:115-001-003)
    抗Flag(Sigma-Aldrich,目录号:F3165)
    抗PKC-θ(Santa Cruz Biotechnology,目录号:sc-1875; BD,Transduction Laboratories TM,目录号:610089)
    抗SUMO1(Santa Cruz Biotechnology,目录号:sc-9060)
  20. Tris(Sangon Biotech,目录号:A600194)
  21. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  22. Nonidet P40(Sangon Biotech,目录号:A600385)
  23. 抑肽酶(EMD Millipore,目录号:616370)
  24. 亮肽素(Calbiochem,目录号:108976)
  25. 苯基甲磺酰基吡咯(PMSF)(Sigma-Aldrich,目录号:P7626)
  26. 焦磷酸四钠十水合物(NaPPi)(Sigma-Aldrich,目录号:S6422)
  27. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:567540)
  28. 蛋白G琼脂糖凝胶(GE Healthcare,目录号:17-0618-01)
  29. 裂解缓冲液(见配方)

设备

  1. 血细胞计数器(Beckman Coulter,型号:Z1 COULTER COUNTER)
  2. 加湿CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Forma TM 3111)
  3. 层流气流生物安全柜(ESCO Micro,型号:Airstream class II)
  4. 离心机(Eppendorf,型号:5417R)

软件

  1. ImageJ软件

程序

  1. 细胞制备和细胞培养
    1. 用500 ml HBSS稀释500 ml血液,在50ml锥形管中小心地将35 ml稀释的血液层加在15 ml Ficoll上。
    2. 在25℃,在没有制动器的摆动转子转子中以350×g离心40分钟。
    3. 吸出上层,使单核细胞层不受干扰。
    4. 小心地转移单核细胞层到一个新的50ml锥形管。用HBSS填充试管,混合,并在25℃下以300×g离心10分钟。
    5. 小心取出上清液。将细胞沉淀重悬于50ml HBSS中,并在25℃下以200×g离心15分钟。
    6. 小心取出上清液。重悬细胞沉淀在10ml含有2mM EDTA和0.5%BSA的PBS中。确定细胞数量。
    7. 在25℃下以300×g离心细胞悬浮液10分钟。完全吸出上清液。
    8. 将细胞沉淀重悬于含有2mM EDTA和0.5%BSA /10μL细胞的80μlPBS中,每10 7个细胞加入20μlCD4微珠。混合并在冰箱中在4℃下孵育15分钟。
    9. 通过加入2ml含有2mM EDTA和0.5%BSA/10 7个细胞的PBS洗涤细胞,并在25℃下以300×g离心10分钟。
    10. 除去上清液并在500μl含有2mM EDTA和0.5%BSA的PBS中再悬浮至10 8个细胞。然后根据CD4微珠数据表进行磁性分离以富集CD4 + T细胞,并且通过单核细胞的CD4 级分保存作为初级抗原 - (APC),其被非常低百分比的CD8 + T细胞污染但不影响以下刺激。
    11. 在补充有10%热灭活的FBS和1%抗生素 - 抗真菌溶液的RPMI-1640培养基中培养CD4 + T细胞或Jurkat T细胞,初级APC或Raji B细胞。
  2. 细胞刺激
    1. 超级抗原(SEE) 刺激
      1. 在有或没有500ng/ml(初级APC)或100ng/ml(初级APCs)的情况下孵育初级APC(1×10 7个)或Raji B细胞(1×10 7个) Raji B)在1.5ml微量离心管中在37℃下在200μl终体积的RPMI-1640培养基(10%FBS)中SEE 30分钟。然后在25℃下以300×g离心细胞3分钟,并用冰冷的PBS洗涤沉淀物以获得SEE负载的APC-SEE或Raji B-SEE细胞。
      2. 用1ml冰冷PBS洗涤初级CD4 +或Jurkat T细胞(1×10 7个/孔)一次,并重悬于200μl终体积的RPMI-1640培养基中,并孵育在冰上15分钟
      3. 将APC-SEE或Raji-B-SEE与T细胞以1:1的比例(在400μl最终体积中)混合,在4℃,300×g下离心1分钟。保持细胞沉淀在培养基中,并立即孵育细胞沉淀1,5和15,30分钟,在37℃。
    2. 抗体刺激
      1. 在200μl无血清RPMI中重悬Jurkat T(1×10 7个)或人原代CD4 +细胞(1×10 7个/sup)。在冰上孵育10分钟。然后加入10μg/ml抗CD3和/或2μg/ml抗CD28单抗,将混合物置于冰上5分钟,然后用山羊抗小鼠IgG(10μg/ml)交联,并立即孵育细胞在37℃下孵育1,5,15,30分钟。
  3. 在350×g离心细胞3分钟,小心地除去上清液,并在120μl裂解缓冲液(Xie等人,2013)中裂解刺激的细胞,其含有20mM (NEM)在冰上10分钟。
  4. 通过离心(8,500×10g,10分钟,4℃)清除裂解物,然后向上清液中加入1%SDS(v/v),并通过在90℃加热10分钟解离免疫沉淀的蛋白质min。
  5. 用裂解缓冲液将步骤4中获得的裂解物稀释10倍,并用抗Flag M2或山羊抗PKC-θAbs分别在4℃下用旋转过夜免疫沉淀Flag标记的PKC-θ或内源性PKC-θ过夜。抗IgG免疫沉淀和来自未刺激细胞裂解物的IPs平行进行作为阴性对照。然后加入30μl蛋白G珠(50%[v/v]的PBS溶液),并在4°C下旋转孵育2-4小时。
  6. 在4℃下以8,000×g离心5分钟,小心除去上清液,通过加入1ml含有20mM NEM的裂解缓冲液并涡旋3-5分钟,彻底洗涤免疫沉淀物。
  7. 重复洗涤步骤另外4次。然后将样品进行SDS-PAGE,并用抗SUMO1(1:1,000)或小鼠抗PKC-θ(1:1,000)Abs进行免疫印迹。典型的免疫印迹显示在代表数据部分(图5)。

数据分析

  1. 为了排除由抗SUMO1抗体检测的非特异性条带,包括来自未刺激细胞裂解物的抗IgG免疫沉淀物(IP)和IP的一组阴性对照应包括在试验实验中。如果控制通道的信号几乎检测不到并且细胞刺激成功,则系统工作。
    1. 使用ImageJ软件分析信号如下:
      点击"文件"和"打开",然后选择图像。
    2. 点击"矩形"工具,出现一个带黄线的框。在信号周围拖动以定义要分析的区域。单击"Ctrl + 1"选择第一行(图1)。


      图1. ImageJ 中的PKC-θ的sumoylated带的选择

    3. 使用箭头键将框移动到下一个车道,而不更改框的大小,单击"Ctrl + 2"选择下一个车道。重复此步骤直到选择所有通道。
    4. 用箭头键移动框到空白区域,点击"Ctrl + 2"选择背景
    5. 点击"Ctrl + 3"绘制车道。一系列图将出现在新窗口中(图2)。


      图2.点击"Ctrl + 3"后的ImageJ窗口

    6. 选择"Wand工具",然后单击每个图的区域(图3)。打开一个新窗口,其中体积数据表示定义的边界内的像素强度。从所有值中减去背景像素强度(使用空白区域的值),以获得相对强度值(图4)。


      图3.选择"魔杖工具"


      图4.分析后的ImageJ界面

    7. 用类似的方法定量PKC-θ印迹的信号
    8. 用其相应的PKC-θ值标准化每个泳道的SUMO1值。

代表数据



图5.人原代CD4 + T细胞中PKC-θ(PKC-θ-SUMO1)的sumoylation的体内测定。使用SEE脉冲的人APC(APC + SEE)以1:1的比例刺激分离的人原代CD4 + T细胞0-30分钟(以上泳道),通过免疫印迹分析(顶部)和没有免疫沉淀的全细胞裂解物(WCL)(底部)免疫沉淀(IP)的蛋白质(图1B),用各种分子的抗体(左边缘)探测。肌动蛋白作为加载对照(Wang等人,2015)。

笔记

  1. 由于多个SUMO附着位点,应当预期蛋白质的更大的表观分子大小。因此,推荐使用较低浓度的丙烯酰胺凝胶(,<8%)。
  2. 重要的是,细胞应该在刺激后尽可能快地裂解,因为sumoylation通常瞬时发生并且容易被SUMO特异性蛋白酶解偶联。此外,推荐在免疫沉淀过程中的裂解和洗涤步骤中在裂解缓冲液中补充20mM的N-乙基马来酰亚胺(这些蛋白酶的化学抑制剂)。
  3. 由于内源性PKC-θ的sumoylated带的信号可能非常弱,膜与抗SUMO1 Ab在4°C过夜孵育和更长的暴露时间,当可视化的免疫印迹信号强烈推荐。

食谱

  1. 裂解缓冲液
    20mM Tris-HCl(pH7.5) 150mM NaCl 5 mM EDTA
    1%Nonidet P40
    10μg/ml抑肽酶
    10μg/ml亮肽素 1mM PMSF
    5 mM NaPPi
    1mM Na 3 VO 4 sub。

致谢

该方案从体内泛素化试验中修改(Wertz等人,2004)。由中国国家自然科学基金(31170846)和中国科学技术部支持(2013CB835300)。

参考文献

  1. Wang,XD,Gong,Y.,Chen,ZL,Gong,BN,Xie,JJ,Zhong,CQ,Wang,QL,Diao,LH,Xu,A.,Han,J.,Altman, Y.(2015)。  TCR诱导的激酶PKC-θ控制T细胞突触组织和T细胞活化。 Nat Immunol 16(11):1195-1203。
  2. Wertz,IE,O'Rourke,KM,Zhou,H.,Eby,M.,Aravind,L.,Seshagiri,S.,Wu,P.,Wiesmann,C.,Baker,R.,Boone,DL,Ma ,A.,Koonin,EV和Dixit,VM(2004)。  430(7000):694-699。
  3. Xie,JJ,Liang,JQ,Diao,LH,Altman,A.和Li,Y。(2013)。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, X., Chen, Z., Wang, Q. and Li, Y. (2016). Assessment of TCR-induced Sumoylation of PKC-θ. Bio-protocol 6(20): e1979. DOI: 10.21769/BioProtoc.1979.
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