Inhibition of PI3K is associated with decreased ERK1/2 and increased p38 phosphorylation. HOKs were pretreated with 25-400 nM Wortmannin for 1 h followed by stimulation with (a,c) thrombin (10 U/ml) or (b,d) trypsin (10 nM). Phospho ERK1/2 at 5 min and phospho p38 at 15 min after PAR1/PAR2 activation were quantified in cell lysate by ELISA. Data are given as percent of unstimulated cells and as means ± S.E.M from two different donors. (p < 0.05, * vs. cells treated with thrombin or trypsin in the absence of inhibitors, # vs. Wortmannin 100 nM+ (thrombin or trypsin)). (e) For immunoblot analysis of phospho-ERK1/2 and phospho-p38, HOKs were preincubated with Wortmannin (400 nM) for 1 h followed by stimulation with thrombin (10 U/ml) or trypsin (10 nM). By using specific antibodies for phosphor-ERK1/2, total ERK1/2, phosphor-p38 and total-p38, phosphorylation of ERK1/2 and p38 was evaluated at 5 min and 15 min, respectively. To test the efficacy of Wortmannin in blocking PI3K/Akt pathway, above sample lysates were analyzed by immunoblotting using antibodies specific for phospho-Akt and total-Akt. GAPDH was used as a control for equal loading of samples.