| Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
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Author:
Date:
2017-05-20
[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for ...
[摘要] 为了实现克氏锥虫内源蛋白的C末端标记，我们使用Cas9 / pTREX-n载体（Lander等，2015）在3'末端插入特异性标签序列（3xHA或3xc-Myc）特定的感兴趣基因（GOI）。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增，并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记，加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M（Oberholzer等，2006）或pMOHX1Tag4H（Lander等，2016b）用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。
【背景】自从CRISPR / ...
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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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| Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination
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Author:
Date:
2015-07-05
[Abstract] Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination ...
[摘要] 泛素化是泛素 - 蛋白酶体通路的第一步，其调节细胞的内环境稳定功能，并且是酶，蛋白质翻译后修饰过程，其中通过一组三种泛素酶将泛素转移到靶蛋白质底物（Weissman et al。，2011; Bhattacharyya et al。，2014; Ristic 等人，2014）。考虑到这一过程的重要性，似乎可能的是，泛素化受到许多因素的严格控制，并且调节机制是蛋白质特异性的。用于检测特异性蛋白泛素化的测定将使我们能够检查因子是否具有调节该蛋白的泛素化的功能。在这里我们描述一个协议，检测人类REST4蛋白在培养细胞中的神经选择性剪接异构体（RE-1沉默转录因子），拮抗REST对神经分化和神经元形成的镇压功能的泛素化状态。使用这个协议，我们显示端粒结合蛋白TRF2通过抑制其泛素化稳定人类REST4的表达。这表明TRF2在神经分化中发挥阳性作用（Ovando-Roche等人，2014）。该方案还可用于检测其他感兴趣的蛋白质的泛素化。
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