![Fig. 4. Detection of MARCKS in platelet extracts and its phosphorylation during PKC activation. / Platelets labeled with [32P]Pi were permeabilized with 15 μM of digitonin in K+-glutamate buffer for 5 minutes. During the last 3 minutes of permeabilization, 100 nM of PMA in 0.05% DMSO (final concentration) or 0.05% DMSO (control) were present in the incubation medium. Reactions were stopped with RIPA buffer containing protease and phosphatase inhibitors. SDS-PAGE was performed on boiled extracts of these preparations to separate heat-stable proteins. Proteins were electrotransferred to nitrocellulose membranes, and autoradiography was first performed. This was followed by Western blotting with a mouse monoclonal antibody (4 μg/mL) raised against the C-terminal domain of human MARCKS. Two bands of 83 and 85 kd were detected by the antibody, which corresponded to 2 phosphorylated protein bands found in the autoradiography. Although PMA increased the phosphorylation of both MARCKS bands, the upper band was heavily phosphorylated, increasing the amount of slow-moving MARCKS (85 kd), as indicated by a much heavier immunoreactivity of this band in the PMA-treated preparations.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/95/3/10.1182_blood.v95.3.894.003k15_894_902/6/bloo00315004w.jpeg?Expires=1724239829&Signature=nQn-9YM0aU7N0qZ7A5Z2TseAAZRtXj54TyxdbgYrqwnltBUoiRGPQuaiEqqfhlWGMTmmGYtk~g5syVG2dBxe~nuS4f9BF8aKPgYppcxsq8wn7m7ZwX9EP0ZDEsrEIV~8Oi~~~12szauTqyy9WWWo3F3PH7r77g-0IAcxkM4uwK3CJba-LNwlWdHj0r5n6ycj-2MDR3cZU9CUMu95b1xD1cPVmNk-HgzTo2lO-cls86rSWSloSVs8OoAEpuKjugK31d-URbi9XRYeiu5DJ8Va0onpNJIcTdq-bTIh1oe1hq2FYtY40XSbEojJ2aM30hkpPI1EDjU0LWqkfcCKHg8YVg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Detection of MARCKS in platelet extracts and its phosphorylation during PKC activation.
Platelets labeled with [32P]Pi were permeabilized with 15 μM of digitonin in K+-glutamate buffer for 5 minutes. During the last 3 minutes of permeabilization, 100 nM of PMA in 0.05% DMSO (final concentration) or 0.05% DMSO (control) were present in the incubation medium. Reactions were stopped with RIPA buffer containing protease and phosphatase inhibitors. SDS-PAGE was performed on boiled extracts of these preparations to separate heat-stable proteins. Proteins were electrotransferred to nitrocellulose membranes, and autoradiography was first performed. This was followed by Western blotting with a mouse monoclonal antibody (4 μg/mL) raised against the C-terminal domain of human MARCKS. Two bands of 83 and 85 kd were detected by the antibody, which corresponded to 2 phosphorylated protein bands found in the autoradiography. Although PMA increased the phosphorylation of both MARCKS bands, the upper band was heavily phosphorylated, increasing the amount of slow-moving MARCKS (85 kd), as indicated by a much heavier immunoreactivity of this band in the PMA-treated preparations.
