| Synaptoneurosome Preparation from C57BL/6 Striata
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Author:
Date:
2016-02-20
[Abstract] Activity-dependent local mRNA translation endows synapses to remodel their structure and function (Bramham and Wells, 2007). This process is tightly controlled by the state of phosphorylation of several components of the translational machinery including initiation factors and ribosomal proteins (Buffington et al., 2014). The present protocol describes a method to prepare striatal synaptoneurosomes, from adult mice, containing both pre- and postsynaptic elements in which the level of synaptic phospho-proteins can be quantified (Biever et al., 2015).
[摘要] 活动依赖的局部mRNA翻译赋予突触重塑其结构和功能（Bramham和Wells，2007）。 该过程由包括起始因子和核糖体蛋白在内的翻译机理的几个组分的磷酸化状态来严格控制（Buffington等，2014）。 本方案描述了一种从成年小鼠制备纹状体synaptoneurosomes的方法，其含有可以量化突触磷酸蛋白水平的突触前和突触后元件（Biever等，2015）。
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| Detection of Phospho-KRAS by Electrophoretic Mobility Change in Human Cell Lines and in Tumor Samples from Nude Mice Grafts
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Author:
Date:
2015-03-20
[Abstract] KRAS is the oncogene most frequently mutated in human solid tumors especially in pancreas, colon, small intestine, biliary tract and lung. We have recently demonstrated that oncogenic KRAS needs S181 phosphorylation to fully display its oncogenic features suggesting its inhibition as a therapeutic treatment against KRAS-driven tumors. Due to the importance to detect KRAS phosphorylation in human tumors and the absence of specific antibodies against phosphorylated KRAS, we developed a new protocol based on the Phos-tag SDS methodology to detect this post-translational modification for KRAS. ...
[摘要] KRAS是在人实体瘤特别是在胰腺，结肠，小肠，胆道和肺中最常突变的癌基因。 我们最近证明，致癌KRAS需要S181磷酸化充分显示其致癌特征，表明其作为对KRAS驱动的肿瘤的治疗性治疗的抑制作用。 由于在人类肿瘤中检测KRAS磷酸化的重要性和不存在针对磷酸化KRAS的特异性抗体，我们开发了基于Phos-tag SDS方法的新方案以检测这种KRAS的翻译后修饰。 Phos标签是特异性结合磷酸化蛋白质的分子，降低其在SDS-PAGE中的迁移速度并允许其从非磷酸化形式分离。
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| Purification of HCV-remodeled and Control ER Membranes
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Author:
Date:
2014-05-20
[Abstract] As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate HCV-remodeled host cell membranes and endoplasmic reticulum (ER) membranes of naïve cells, by using a functional NS4B HA-tagged subgenomic replicon and a C-terminally HA-tagged calnexin-overexpressing cell line, respectively. Post-nuclear whole cell membrane ...
[摘要] 至于所有正链RNA病毒，丙型肝炎病毒（HCV）RNA复制与称为病毒复制工厂的重排宿主细胞膜紧密相关。 然而，到目前为止对病毒复制工厂的病毒和细胞成分了解甚少。 在这里，我们描述一个协议，通过使用功能NS4B HA标记亚基因组复制子和C末端HA标记calnexin过表达细胞系，特异性隔离HCV重塑的宿主细胞膜和内质网（ER）膜的幼稚细胞， 分别。 首先通过密度梯度离心富集核后全细胞膜级分，随后通过HA特异性亲和标签纯化。 在天然条件下洗脱时，纯化的样品可进行多种生物化学和功能测定。
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