Figure 7
Mitogen induce activation of CREB is serine-133 phosphorylation dependent but CRTC independent. VSMC were transfected with CREB-LUC and serum starved for 24 hours. Cells were stimulated with 5% serum or 25 ng/ml PDGFBB for the indicated times and luciferase activity quantified (n = 3; A). VSMC serum starved for 4 h were stimulated for 5, 15, 30, 60 or 120 min with 5% FCS or 25 ng/ml PDGFBB (B) or pre-treated with UO126 (UO, 10 µM), MK-2206 (MK, 10 µM), LY294002 (LY, 10 µM), Bisindolylmaleimide I (BIS1, 5 µM), SB 203580 (SB, 10 µM) or SP600125 (SP, 10 µM) and then stimulated with PDGF for 30 min (C). Phosphorylated CREB was detected in cell lysates by western blotting with a pCREB (S133) antibody, membranes were reprobed with a total CREB antibody. Phospho-CREB bands were quantified by densitometry and expressed relative to the maximum value in each individual experiment. VSMC were transfected with GAL4-NLUC together with either a GAL4, GAL4-CREBwild-type or GAL4-CREBS133A expression vector and serum starved for 24 hours before stimulation with 5% serum for 4 hours (n = 5; D). VSMC (n = 6) serum starved for 4 h were treated for 20 or 60 min with 5% FCS or 25 ng/ml PDGF. Cells were subjected to fractionation and nuclear extracts analysed by Western blotting and densitometry for CRTC2 and CRTC3 (E). *Indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001 relative to non-stimulated; one-way repeated measures ANOVA with Student Newman Keuls post-hoc tests.
