Fig. 4.

Phosphoinositide 3-kinase (PI3K) activation inhibits IL-1β-induced upregulation of RGS4 in rabbit colonic SMCs. A: IL-1β induces the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β). Cultured and serum-starved SMCs were treated with IL-1β (10 ng/ml) for indicated time periods followed by Western blot analysis with anti-p-Akt or anti-p-GSK3β antibodies. B–D: PI3K inhibition enhances IL-1β-induced upregulation of RGS4 mRNA and protein expression. Confluent colonic SMCs were serum starved for 24 h and pretreated with DMSO, the PI3K inhibitor LY-294002 (LY; 10 μM), or Src kinase inhibitor I (10 μM) for 1 h before an exposure to IL-1β (10 ng/ml) for 3 h (B) and 3 days (B–D). Expression levels of RGS4 mRNA were determined by conventional RT-PCR (B) and real-time RT-PCR using GAPDH for normalization (C). The expression of RGS4 protein was measured by Western blot analysis (D). The number under each blot indicates the relative fold of optical density compared with the corresponding control.