| In Gel Kinase Assay
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Author:
Date:
2017-03-05
[Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, ...
[摘要] 正常发育和体内平衡需要细胞和组织中蛋白质磷酸化的适时时空调节。我们提出方案“凝胶激酶测定”，其可用于粗蛋白质提取物的蛋白激酶活性测量。我们已经成功地使用“凝胶激酶测定”方案来证明在ABA受体（PYR / PYL / RCAR-ABA受体）的PYRABACTIN RESISTANCE1 / PYR1-样/调节组分中的拟南芥线条pyr / pyl112458 ）在ABA介导的SnRK2.2，SnRK2.3和OST1 / SnRK2.6的活化中受损，多达三重突变体snrk2.2 / 2.3 / 2.6 （Gonzalez-Guzman等人，2012）。
背景 植物激素脱落酸（ABA）是涉及植物生长发育以及植物对非生物和生物胁迫的反应的关键信号。 ABA感知和信号通路由ABA受体（PYR / PYL / RCAR-ABA受体）的PYRABACTIN RESISTANCE1 / PYR1-调节组分，PP2C磷酸酶和SnRK2s激酶组成（在Antoni等人， ，，2011）。模块受体-ABA-磷酸酶通过调节ABA激活的SnRK2而以配体依赖的方式控制磷酸化信号级联。反过来，SnRK2s激酶使细胞核和细胞质中的无数效应物从转录因子（例如，ABFs）到离子通道（例如）磷酸化， ...
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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 ...
[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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| Substrate Specificity of Recombinant Ser/Thr Protein Kinase
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Author:
Date:
2015-03-20
[Abstract] Protein kinases are enzymes that phosphorylate proteins in a cell. Determination of kinase activity in reactions of phosphorylation is a very convenient way for a biochemical characterization of this group of enzymes. Here we describe a method to determine the activity of a recombinant Ser/Thr protein kinase using as a possible substrate MBP, H1, and BSA.
[摘要] 蛋白激酶是磷酸化细胞中蛋白质的酶。 磷酸化反应中激酶活性的测定是这组酶的生物化学表征的非常方便的方法。 在这里，我们描述了一种使用可能的底物MBP，H1和BSA确定重组Ser/Thr蛋白激酶活性的方法。
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