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Author:
Date:
2017-07-05
[Abstract] Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal ...
[摘要] Cdk5活性受两种激活蛋白p35和p39（Tsai et al。，1994; Zheng et al。，1998; Humbert等人）的量的调节，2000）。 p35-Cdk5和p39-Cdk5复合物对盐和洗涤剂浓度的敏感性不同（Hisanaga和Saito，2003; Sato et al。，2007; Yamada等人， 2007; Asada 等人，2008）。 Cdk5激活可以通过Cdk5与其结合的激活剂的免疫沉淀直接测量，随后进行Cdk5激酶测定。在该方案中，用于细胞裂解和免疫沉淀的缓冲液旨在保持p35-和p39-Cdk5复合物以评估总Cdk5活性。裂解细胞，并在核后上清液中测定蛋白浓度。 Cdk5在实验组之间从等量的总蛋白免疫沉淀。然后进行洗涤以除去外来蛋白质并平衡激酶缓冲液中的Cdk5-活化剂复合物。然后将Cdk5与组蛋白H1孵育，组蛋白H1是Cdk5和[γ- 32 P] ATP在体外成功建立的靶标。反应通过SDS-PAGE解析并转移到膜上，用于可视化H1磷酸化和免疫沉淀的Cdk5水平的免疫印迹。我们已经使用该测定来建立p39作为少突神经胶质谱系中Cdk5的主要活化剂。然而，该测定法适用于对裂解条件进行适当调整的其它细胞谱系或组织。
【背景】虽然Cdk5通常与神经元功能相关，但最近的工作已经证明Cdk5也可以调节少突胶质细胞祖细胞（OPC）的发育（Tang等人，1998; ...
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Author:
Date:
2012-11-05
[Abstract] This is a general protocol for the isolation and maintenance of primary MPM cultures as a tool for the identification of Tumor Initiating Cells and early progenitor-targeting drugs (Cioce et al., 2010). Primary cultures can be propagated efficiently for 8-12 weeks and xenotransplanted in NOD/SCID mice while retaining the histofeatures of the originating tumor (Canino et al., 2012). The protocol is suitable for both MPM solid specimens and pleural effusion. For increased clarity, initially two separate sections addressing the isolation of MPM cells from solid tumors and pleural effusions are here provided.
[摘要] 这是用于分离和维持初级MPM培养物的一般方案，作为鉴定肿瘤启动细胞和早期祖细胞靶向药物的工具（Cioce等人，2010）。 原代培养物可以有效繁殖8-12周，并且在NOD/SCID小鼠中异种移植，同时保留起源肿瘤的组织特征（Canino等人，2012）。 该方案适用于MPM固体标本和胸腔积液。 为了增加清晰度，最初提供了两个分开的部分，其涉及从实体瘤和胸腔积液中分离MPM细胞。
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