| Cell Surface Protein Detection to Assess Receptor Internalization
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Author:
Date:
2016-10-20
[Abstract] The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the ...
[摘要] 膜受体在暴露于不同的刺激剂/抑制剂时的迁移是非常重要的。其中，膜受体的内化影响其对配体的可及性和对环境线索的细胞应答性。在实验上，受体内化可用作其活化的量度。在我们的研究中，我们采用这种方法来探讨神经肽Y（NPY）的七个跨膜结构域受体，Y5R和脑源性神经营养因子（BDNF），TrkB的酪氨酸激酶受体之间的串扰。为此，我们测量了用TrkB配体BDNF刺激后Y5R的内化。在用BDNF处理后，将细胞暴露于选择性标记表面蛋白的不透膜的生物素化试剂。随后，生物素化的膜蛋白在具有抗生物素蛋白树脂的柱上亲和纯化，并通过蛋白质印迹分析。存在于对照和配体处理的细胞的细胞表面上的受体部分的差异用作其内化和对特定刺激的反应的量度。
[Backg 回合] 可以使用两种主要策略 - 显微镜和生物化学来测量响应外部刺激的细胞膜受体内化。最常见的方法是使用显微镜 - ...
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| Analysis of Protein Stability by the Cycloheximide Chase Assay
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Author:
Shih-Han Kao, Wen-Lung Wang, Chi-Yuan Chen, Yih-Leong Chang, Yi-Ying Wu, Yi-Ting Wang, Shu-Ping Wang, Alexey I Nesvizhskii, Yu-Ju Chen, Tse-Ming Hong and Pan-Chyr Yang,
Date:
2015-01-05
[Abstract] Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.
[摘要] 真核细胞中蛋白质稳定性的比较已经通过放线菌酮实现，放线菌酮是蛋白质生物合成的抑制剂，由于其在翻译延伸中的预防。 它在确定给定蛋白质的半衰期方面广泛用于细胞生物学，并且已在癌症研究中获得广泛应用。 在这里，我们在我们的实验室使用肺腺癌细胞系CL1-5作为模型呈现完整的放线菌酮追踪测定法。
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