Fig. 4. Effects of pertussis toxin and LY294002 on phosphorylation of Akt and expression of CCL11 induced by thrombin. (A) HAo-SMCs were stimulated with thrombin for 5 min after incubation with or without pertussis toxin (100 ng/ml) and LY294002 (10 µM) for 2 h. An equal amount of protein was subjected to Western blot analysis with antibodies against α-tubulin and phosphorylated and unphosphorylated forms of Akt. The mean band intensity of p-Akt was normalized to that of Akt. (B, C) HAoSMCs were incubated with or without pertussis toxin (100 ng/ml) and LY294002 (10 µM) for 2 h followed by stimulation with thrombin for 9 h. The levels of CCL11 transcripts were determined by realtime PCR (B), and the levels of CCL11 protein secreted into culture media were determined by ELISA (C). Data are expressed as mean±SD (n=3 replicates for each group). ^***p<0.001 vs. control; ^###p<0.001 vs. thrombin.