| MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation
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Author:
Date:
2016-12-05
[Abstract] Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of ...
[摘要] 免疫沉淀（IP）代表广泛使用的生物化学方法，以利用特异性识别特定靶分子的抗体从复杂混合物中分离特异性蛋白质。该程序是非常通用的，可以应用于集中特定蛋白质，识别与其复合的相互作用的配偶体或检测翻译后修饰。有丝分裂蛋白单克隆2（MPM-2）是最初针对同步有丝分裂HeLa细胞的提取物产生的抗体，以鉴定选择性存在于有丝分裂中的蛋白质，而不是在间期细胞中（Davis等，1983）。 MPM-2识别磷酸化的丝氨酸或苏氨酸残基，随后是脯氨酸（pS / T-P），由脯氨酸指导的蛋白激酶和磷酸酶的共同作用产生的共有表位（Lu等，2002）。这些可逆磷酸化事件已经出现以通过促进目标上的构象变化来控制各种细胞过程，这不仅仅是由于磷酸化事件本身。这些基序一旦被磷酸化，就能够招募Pin1（肽基 - 脯氨酰异构酶NIMA相互作用蛋白1）（Lu等人，1996; Lu和Zhou，2007），这是一个促进肽键上的顺式/反式异构化反应的伴侣，在基础功能不同的构象之间切换底层（Lu，2004; Wulf等人，2005）。该方案描述了使用支架分子突触后密度蛋白-95（PSD-95）（Chen等人，2005），神经元Pin1靶（Antonelli等，2016）作为示例的基于MPM-2的免疫沉淀策略以说明详细的程序。
【背景】由MPM-2抗体识别的抗原的鉴定代表了发现经过磷酸化脯氨酰异构化调控机制的靶分子的有用起点。 ...
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| Assessment of TCR-induced Sumoylation of PKC-θ
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Author:
Date:
2016-10-20
[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.
[摘要] 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; ...
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| PKC-θ in vitro Kinase Activity Assay
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Author:
Date:
2016-10-20
[Abstract] 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. In T cells, TCR and CD28 stimulation-induced activation of PKC-θ is the integrated result of diacylglycerol-mediated membrane recruitment, GLK-mediated phosphorylation at activation loop, CD28, Lck, and sumoylation-mediated central immunological synapse localization (Wang et al., 2015; Monks et al., 1997; Kong et al., 2011; Isakov and Altman, 2012; Chuang et al., 2011). ...
[摘要] Sumoylation控制许多细胞过程。蛋白激酶C-θ（PKC-θ）是激酶的Ca 2+超家族PKC亚家族的成员，通过介导T细胞抗原受体（TCR）作为T细胞活化的调节剂， - 和共受体CD28诱导的转录因子NF-κB和AP-1的活化，以及较小程度的NFAT，以及随后的白细胞介素2（IL-2）产生和T细胞增殖。我们最近证明了TCR诱导的PKC-θ的sumoylation是其在T细胞中的功能所必需的（Wang等人，2015）。在这里我们描述分析TCR诱导的过表达或内源性PKC-θ的sumoylation的方法，这是通过免疫沉淀PKC-θ进行，然后用抗SUMO1抗体进行免疫印迹。
[背景] ...
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