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Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cellsстатья Исследовательская статья
Информация о цитировании статьи получена из
Web of Science,
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 15 апреля 2020 г.
- Авторы: Kazakova O.A., Khapchaev A.Y., Ragimov A.A., Salimov E.L., Shirinsky V.P.
- Журнал: Biochemistry (Moscow)
- Том: 84
- Номер: 1
- Год издания: 2019
- Издательство: Pleiades Publishing, Ltd
- Местоположение издательства: Road Town, United Kingdom
- Первая страница: 11
- Последняя страница: 19
- DOI: 10.1134/S0006297919010024
- Аннотация: Myosin II is the main molecular motor to drive actomyosin-dependent motility events in cells. Myosin regulatory light chain (RLC) phosphorylation 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 exerts additional stimulatory effect on 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 for measurements in non-muscle cells, which have low myosin II content and are usually available at lower quantities than muscle tissue. Therefore, it becomes a formidable task to conduct a dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells due to high input of cells required. Employment of phosphosite-specific antibodies increases the sensitivity of detection but allows determining only relative amount of phosphate incorporation in RLC at specific sites; thus, an ambiguity arises with regard to physiologic relevancy of the observed changes in RLC phosphorylation. As an approach for quantitative RLC phosphorylation measurements in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in-vitro phosphorylated RLC species and utilized a conventional SDS-PAGE and Western blot with phospho-specific RLC antibodies. In this report, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation changes in human endothelial cells in response to natural agonists, either thrombin or insulin, and in intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for myosin RLC phosphorylation measurement in non-muscle cells.
- Добавил в систему: Хапчаев Аскер Юсуфович