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首页> 外文期刊>Biochemistry >Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cells
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Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cells

机译：基于Western印迹的肌蛋白II调节轻链磷酸盐的定量测量少量非肌肉细胞

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摘要

Myosin II is the main molecular motor in the actomyosin-dependent motility in cells. Phosphorylation of the myosin regulatory light chain (RLC) at Ser19 is a prerequisite for smooth muscle/non-muscle myosin II activation and serves as a biochemical equivalent of myosin II activity. Simultaneous phosphorylation at Thr18 further promotes the myosin II ATPase activity. A number of methods have been developed to measure myosin RLC phosphorylation at Ser19 or di-phosphorylation at Thr18/Ser19. While these methods are straightforward and robust in myosin-rich muscle tissues, they demonstrate limited applicability in non-muscle cells that have low myosin II content and are usually available in lesser amounts than muscle tissue. Because of this, dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells is difficult and requires large number of cells. The use of phospho-specific antibodies increases detection sensitivity but allows estimation of only relative levels of RLC phosphorylation at specific residues, which makes it difficult to estimate the physiologic relevancy of the observed changes in RLC phosphorylation. To measure RLC phosphorylation in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in vitro phosphorylated RLC in standard SDS-PAGE and Western blot procedures with phospho-specific RLC antibodies. Here, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation in endothelial cells in response to natural agonists (thrombin or insulin) and intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for measuring myosin RLC phosphorylation in non-muscle cells.
机译：肌球蛋白II是细胞中依赖于侵袭性的主要分子电机。 Ser19在Ser19的肌霉素调节轻链（RLC）的磷酸化是平滑肌/非肌肉肌蛋白II活化的先决条件，用作肌球蛋白II活性的生物化学等同物。 THR18的同时磷酸化进一步促进肌球蛋白II ATP酶活性。已经开发了许多方法来测量SER19或THR18 / SER19的SER19或二磷酸化的肌球蛋白RLC磷酸化。虽然这些方法在富含肌菌素的肌肉组织中具有直截了当且稳健的肌肉组织，但它们在具有低肌蛋白II含量的非肌肉细胞中表现出有限的适用性，并且通常以较小的量可用于肌肉组织。因此，在多个非肌肉细胞的多个样品中对RLC磷酸化的动态分析难以达到大量的细胞。磷酸特异性抗体的使用增加了检测灵敏度，但允许估计特定残基的RLC磷酸化的相对水平，这使得难以估计所观察到的RLC磷酸化变化的生理相关性。为了测量少量非肌肉细胞的RLC磷酸化，我们在标准SDS-PAGE和Western印迹程序中使用了非磷酸化和体外磷酸化RLC的外部校准标准用磷酸特异性RLC抗体。在这里，我们详细描述了该方法，并证明其在响应于天然激动剂（凝血酶或胰岛素）和完整的人血小板和完整的人血小板中的内皮细胞中肌球蛋白RLC磷酸化的定量测量。我们讨论了所提出的方法对非肌肉细胞中肌蛋白RLC磷酸化的其他方法的优点和局限性。

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