| 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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| In vitro Assessment of Immunological Synapse Formation by Flow Cytometry
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Author:
Date:
2016-03-20
[Abstract] In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical methods that directly measure the number of conjugates as a result of immunological synapse formation between T cells and superantigen-loaded B cells or between cytotoxic T cells and antigen-loaded target cells by dual-color flow cytometry.
[摘要] 在适应性免疫系统中，T细胞和抗原呈递细胞（树突细胞，B细胞和巨噬细胞）或靶细胞（肿瘤细胞和病毒感染细胞）之间的免疫突触的形成对通过细胞因子执行T细胞免疫应答至关重要 分泌或直接杀死活动。 在这里，我们描述了通过双色流式细胞术直接测量T细胞和加载超抗原的B细胞之间或细胞毒性T细胞和抗原负载的靶细胞之间的免疫突触形成的结合物数量的实际方法。
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| Flow Cytometric Analysis of Calcium Influx Assay in T cells
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Author:
Date:
2013-09-20
[Abstract] Calcium influx is one of the key signaling events upon stimulation of T cell receptors (TCR) and plays an important role for T cell activation, proliferation, and differentiation. Phorbol myristate acetate (PMA) and calcium ionophore ionomycin are commonly utilized as stimulants in a variety of immunologic assays including T cell activation. PMA is a protein kinase C (PKC) activator, resulting in the activation of Ras, a small GPTase. When PMA and ionomycin are used together, TCR signaling downstream of PKC and Ras can be activated without activation of TCR-triggerd signaling events. This protocol describes the flow cytometry analysis of intracellular calcium influx in T cells stimulated with PMA and ionomycin.
[摘要] 钙流入是刺激T细胞受体（TCR）的关键信号事件之一，并且对T细胞活化，增殖和分化起重要作用。 佛波醇肉豆蔻酸乙酸酯（PMA）和钙离子载体离子霉素通常在多种免疫测定（包括T细胞活化）中用作刺激剂。 PMA是蛋白激酶C（PKC）激活剂，导致Ras（一种小GPTase）的活化。 当PMA和离子霉素一起使用时，PKC和Ras下游的TCR信号传导可以在没有激活TCR-触发信号事件的情况下被激活。 该协议描述了用PMA和离子霉素刺激的T细胞中的细胞内钙流入的流式细胞术分析。
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