| Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses
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Author:
Date:
2017-12-05
[Abstract] Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein interactions (Duggal and Emerman, 2012). The potency of the IFN signaling response determines the outcome of TBFV infection (Best, 2017; Carletti et al., 2017). Interestingly, data from our lab show that TBFV replication is significantly ...
[摘要] 蜱传黄热病病毒（TBFV）的细胞感染导致干扰素（IFN）信号传导途径的激活和随后称为IFN刺激基因（ISG）（Schoggins等人，2011）的众多基因的上调。许多ISG通过蛋白质 - 蛋白质相互作用以广泛或特定的方式起作用来防止病毒发病（Duggal和Emerman，2012）。 IFN信号反应的效力决定了TBFV感染的结果（Best，2016; Carletti等人，2017）。有趣的是，我们实验室的数据显示TBFV复制在储库物种Peromyscus leucopus的细胞中显着受到限制，从而表明有效的抗病毒应答（Izuogu等人，2017）。我们评估干扰素信号对抗性的相对贡献。通过敲低IFN反应途径中的主要转录因子来抑制白血病。信号转导和转录激活因子1（STAT1）是专门针对在P。 leucopus细胞通过shRNA技术。我们进一步测试了基因敲低对细胞对IFN反应和限制病毒复制的能力的影响;结果表明当STAT1表达被改变时，leucopus细胞对IFN刺激的反应降低，并且对TBFV复制显着更敏感。
【背景】IFN信号是抵抗侵入宿主细胞的黄病毒的第一道防线（Robertson等人，2009; Lazear和Diamond，2015）。通过模式识别受体（PRR）检测与病毒颗粒相关的分子标记，然后通过转录因子引发下游信号从细胞释放1型IFN（Kawai ...
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| Analyze p53 degradation by 35S p53 Pulse Chase Analysis
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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 ...
[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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