STA₂-induced phosphorylation of CPI, MBS at Thr-696, and MLC₂₀ at Ser-19 in intact platelets.

Human platelets were stimulated with 0.5 μM STA₂ at 37°C without stirring for 0, 15, 30, 45, 60, and 120 seconds, as indicated, and whole platelet lysates originating from 30 μL of platelet suspension were resolved by SDS-PAGE. This was followed by immunoblotting with respective antibodies, including anti-MBS, anti–pThr695-MBS, anti–CPI-17, anti–pThr38–CPI-17, anti-MLC and anti–pSer19-MLC₂₀. (A) Paired sets of representative immunoblots. (B) The extent of STA₂-induced phosphorylation of CPI (▴), MBS at Thr-696 (■), or MLC₂₀ at Ser-19 (●). Results are 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 that of each total protein. Similar results were obtained in 3 other experiments using different donor platelets.