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. 2009 Feb 13;284(7):4052–4061. doi: 10.1074/jbc.M805032200

FIGURE 3.

FIGURE 3.

A–C, inhibition of PI3K impairs thrombin-induced p70 S6 kinase phosphorylation. Confluent (A) HUVEC or (B) HLMVEC monolayers were pretreated with LY294002 (50 μm) prior to challenge with thrombin (5 units/ml) for the indicated time periods. C, confluent HUVEC monolayers were pretreated with wortmannin (50 nm) prior to challenge with thrombin (5 units/ml) for the indicated time periods. Total cell lysates were immunoblotted with an anti-phospho-p70 S6K (Thr421/Ser424) antibody. Total p70 S6K levels were used to monitor loading. The bar graph represents the effect of wortmannin on thrombin-induced p70 S6K phosphorylation. p70 S6K phosphorylation normalized to total p70 S6K is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3 for each condition). *, p < 0.05 compared with untreated control; #, p < 0.05 compared with thrombin-stimulated control. D, loss of PI3K prevents thrombin-induced p70 S6 kinase phosphorylation. p85α+/+/β+/+ and p85α–/–/β–/– MEFs were challenged with thrombin (5 units/ml) for 5 min. Total cell lysates were immunoblotted with an anti-phospho-p70 S6K (Thr421/Ser424) antibody. Total p70 S6K levels were used to monitor loading. Results are representative of two to three separate experiments.