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Author:
Date:
2015-08-05
[Abstract] Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with 32P-orthophosphate for 14 h, and then lysed. The 32Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the 32Pi-labeled immunoprecipitated C ...
[摘要] 在HBV C蛋白的羧基末端中的七个丝氨酸和一个苏氨酸中，除了一个（丝氨酸183）外，所有其它丝氨酸出现在RxxS/T共有磷酸受体基序的上下文中，并且还与其他共有基序重叠，例如S/TP，RS ，SPRRR，RRRS/T或RRxS/T，表明各种细胞激酶磷酸化这些残基。 为了确定HBV C蛋白的苏氨酸和/或丝氨酸（丝氨酸157,164,170,172,178和180以及苏氨酸162，adw亚型）是否确实是细胞中的磷酸受体残基，Huh7用一系列C- 蛋白表达突变体，用正磷酸32P标记14小时，然后裂解。 用抗HBc抗体免疫沉淀 32 P标记的裂解物，并通过放射自显影检测32 P标记的免疫沉淀的C蛋白。
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Author:
Date:
2012-11-05
[Abstract] p53, is known as the guardian of the genome and as such requires exquisite regulation not only of its abundance but also its activity. The abundance of p53 can be modulated at the level of transcription, translation, and also via its degradation.
This protocol involves 35S metabolic labelling of newly synthesized proteins followed by a period of chase with "cold" media. Samples are harvested and p53 immunopreciptiated, separated by SDS PAGE and the levels of 35S labelled p53 determined. By comparing the level of 35S p53 at 0 h to those "chased" with cold media (e.g. 60 min) provides an indication of the rate of p53 turnover.
[摘要] p53，被称为基因组的守护者，因此需要精细的调节不仅其丰度，而且其活性。 p53的丰度可以在转录，翻译以及通过其降解的水平上调节。
该方案涉及新合成的蛋白质的35S代谢标记，随后用"冷"培养基追踪一段时间 。 收获样品，并通过SDS PAGE分离p53免疫沉淀，并测定35S标记的p53的水平。 通过将0h时35S p53的水平与用冷培养基（例如60分钟）的"追踪"水平进行比较提供了p53周转速率的指示。
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