Activation of p38 MAPK in HUVECs stimulated by thrombin: a time course.

(A) HUVECs were unstimulated or stimulated with 8 U/mL thrombin for 1, 3, 5, or 15 minutes. HUVEC cell lysates were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. Native and phosphorylated p38 MAPKs were analyzed using specific anti-p38 (lane 1) and antiphosphorylated (phospho-) p38 antibodies (lane 2). After p38 MAPK immunoprecipitation from thrombin-activated HUVEC cell lysates, the kinase activity was tested in an in vitro assay using ATF-2 as a substrate and antiphosphorylated ATF-2 fusion protein antibodies (lane 3). (B) Schematic representation of thrombin-induced p38 phosphorylation kinetics (fold activation relative to unstimulated cells is indicated, *P < .05, **P < .01, n = 3). The phospho-p38 index was calculated for each condition as follows: phospho-p38 concentration/native p38 concentration, each concentration being determined by comparing each band density using a gel analyzer.