Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy |
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Authors: | Kiran Bhadriraju John T Elliott My Nguyen Anne L Plant |
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Institution: | (1) SAIC, Arlington, VA, USA;(2) Cell and Tissue Measurements Group, Biochemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, MD, USA;(3) American University, Washington, DC, USA |
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Abstract: | Background In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the
formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs
in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible
for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed
in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the
phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a
reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells.
This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation
in vascular smooth muscle cells. |
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