| Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
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Author:
Date:
2017-06-05
[Abstract] The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to ...
[摘要] 消除HIV-1患者的主要障碍是后整合延迟（Finzi等人，1999）。抗逆转录病毒治疗仅针对主动复制病毒，而具有低转录活性或无转录活性的潜伏感染仍未得到治疗（Sedaghat等人，2007）。为了消除病毒性水库，一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期（Deeks，2012）。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制，并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近，许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端（BET）家族的成员的治疗潜力（Filippakopoulos等人，2010; Dawson等人＆ / em>，2011; Delmore等人，2011），其包括BRD2，BRB3，BRD4和BRDT。小分子BET抑制剂，例如JQ1（Filippakopoulos et al。，2010; Delmore等人，2011），I-BET（Nicodeme等人＆lt; / ...
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| In vitro Deneddylation Assay
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Author:
Date:
2016-03-20
[Abstract] Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein ...
[摘要] Nedd8是共价连接到作为cullin-RING连接酶（CRL）的一部分的cullin蛋白的保守赖氨酸残基的小的遍在蛋白样蛋白（9kDa）。 CRL是对泛素 - 蛋白酶体途径（UPP）中的蛋白质泛素化重要的主要E3连接酶。 CRL的活性通过脱甲基化（CulA-N8，〜98kDa）和脱甲基化（CulA〜89kDa）的循环调节。 COP9信号体（CSN）和丁二酸酶A（DenA）能够切割Nedd8和CullinA之间的异肽键。与单一蛋白DenA相反，CSN是八亚基多蛋白复合物。将不同的构巢曲霉csn 缺失菌株的蛋白质粗提物与从大肠杆菌（大肠杆菌）表达和纯化的重组CSN亚基混合。使用抗CulA或抗Nedd8抗体的Western杂交实验可以显示Nedd化化合物与Denedd化CulA的比率。使用deneddylation测定，我们可以显示CsnE是在体外连接7-亚基预组装的CSN的最后一个亚基，然后CSN可以通过金属蛋白酶亚基CsnE执行cullin deneddylation。该测定法是一种快速且非昂贵的方法，其显现了用于脱蛋白的蛋白质的酶活性。它还可用于测试除去来自构巢曲霉（构巢曲霉）或其他生物体中的底物的翻译后修饰的其它藻肽的活性。
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| Cell Surface Protein Biotinylation and Analysis
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Author:
Date:
2013-08-20
[Abstract] A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a ...
[摘要] 特异性分离和定量细胞表面膜中的蛋白质的好方法是利用生物素化技术。 它包括在裂解细胞之前用生物素试剂标记细胞表面蛋白，并通过NeutrAvidin下拉分离这些标记的蛋白。 然后，将样品进行SDS-PAGE分离，转移至PVDF膜并用特异性抗体探测。 细胞表面表达的定量通过密度测量对应于感兴趣的蛋白质的条带并随后通过膜蛋白质（作为对照）标准化来完成。
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