![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=1735503843&Signature=VIeMramV3j-GZB6VbQ~uP3x2tAqTt8ae9n~VMHJUa9nRJFtoJdk23zLtbalwpMgFi17lWfXroBtIE8J9xXzguzBtukoygNiGJJrIRnNGRxFNeH9ItKE9WP0ZYCCBQEkjkZ0Gp0Jv9euXSMzWJVIGgwWf3qyv4o7KmR5Dfqh2r0jXxjgnILXzlknhpUHFy~DnZf-Ut6--5I5iD9-86mc9wLem25wRyYQXaHAvwdM0Qsql~jsdJ8Boa9UG6kTFtPhkQIDoR6j2RlJg4hLcs400xDJfBYE29GCIE07fwqM52P45nTn3RhqJKjk8U~PIw-2A0Qlw~VnvMCxc7fMFH~8ggw__&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.
