评估TCR诱导的PKC-θ类泛素化（Sumoylation）修饰

Xudong  Wang; Zhilong Chen; Qilong Wang; Yingqiu Li

doi:10.21769/BioProtoc.1979

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Peer-reviewed

Assessment of TCR-induced Sumoylation of PKC-θ

评估TCR诱导的PKC-θ类泛素化（Sumoylation）修饰

Xudong Wang

ZC Zhilong Chen

QW Qilong Wang

YL Yingqiu Li email

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1979 浏览次数: 7805

评审: Ivan ZanoniMichael EnosMareta Ruseva

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Cover of Nature Immunology, featuring study using the protocol.

Oct 2015

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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

Corning 50 ml conical tube (Corning, catalog number: 430829 )
1.5 ml microcentrifuge tube (Corning, Axygen^®, catalog number: MCT-150-C )
MACS columns (Miltenyi Biotec, catalog number: 130-042-401 )
MACS separators (Miltenyi Biotec, catalog number: 130-042-501 )
Raji B cells (ATCC, catalog number: CCL86 )
Jurkat T cells, Clone E6.1 (ATCC, catalog number: TIB-152 )
Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175095 )
Ficoll (Tbdscience, catalog number: HY2015 )
Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sangon Biotech, catalog number: A610185 )
Bovine serum albumin (BSA)
CD4 microbeads (Miltenyi Biotec, catalog number: 130-045-101 )
Superantigen SEE (Toxin Technology, catalog number: ET404 )
RPMI-1640 medium (GE Healthcare, HyClone^TM, catalog number: SH30027.01 )
Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
Antibiotic-antimycotic solution 100x (Thermo Fisher Scientific, Gibco^TM, catalog number: 15240062 )
N-ethylmaleimide (Sigma-Aldrich, catalog number: E1271-5G )
Sodium dodecyl sulfate (SDS) (Sangon Biotech, catalog number: A600485 )
Antibodies
Anti-CD3 (affymetrix, eBioscience, catalog number: 16-0037-85 )
Anti-CD28 (affymetrix, eBioscience, 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 Laboratories^TM, catalog number: 610089 )
Anti-SUMO1 (Santa Cruz Biotechnology, catalog number: sc-9060 )
Tris (Sangon Biotech, catalog number: A600194 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
Nonidet P40 (Sangon Biotech, catalog number: A600385 )
Aprotinin (EMD Millipore, catalog number: 616370 )
Leupeptin (Calbiochem, catalog number: 108976 )
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Sodium pyrophosphate tetrabasic decahydrate (NaPPi) (Sigma-Aldrich, catalog number: S6422 )
Sodium orthovanadate (Na₃VO₄) (Sigma-Aldrich, catalog number: 567540 )
Protein G sepharose (GE Healthcare, catalog number: 17-0618-01 )
Lysis buffer (see Recipes)

Equipment

Haemocytometer (Beckman Coulter, model: Z1 COULTER COUNTER )
Humidified CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM 3111 )
Laminar air flow bio-safety cabinet (ESCO Micro, model: Airstream class II )
Centrifuge (Eppendorf, model: 5417R )

Software

ImageJ software

Procedure

English

中文翻译

文章信息

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如何引用

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.