PMA-induced phosphorylation of CPI and MLC₂₀at Ser-19 in intact platelets.

Human platelets were stimulated with 100 nM PMA for the indicated time at 37°C under conditions of nonstirring, and whole platelet lysates originating from 30 μL of platelet suspension were resolved by SDS-PAGE. This was followed by immunoblotting with anti–pSer19-MLC₂₀ and anti–pThr38–CPI-17 antibodies. Results were expressed as fold increase in phosphorylation of each protein relative to the level at 0 second. Densities of the immunoreactive bands determined with each antiphosphoprotein antibody were normalized according to total of each protein. Similar results were obtained in 3 other experiments using different donor platelets.