Figure 7.

p38 MAPK and protein phosphatase (PP)5 pathways distinctively modulate GR-mediated transactivation activities. (A) Cells were transfected for 48 hours with 100 nM of p38 siRNA and/or PP5 siRNA or control siRNA before FP (100 nM) was added for 2 hours. Cells were lysed, and whole cell lysate extracts were prepared and assayed for S203, S211, S226, GR, p38 MAPK, PP5, and GAPDH by immunoblot analysis. Results are representative of three separate blots. (B) Top: Cells were transfected for 48 hours with 2 μg luciferase reporter plasmid driven by GRE motifs and 1 μg of β-galactosidase vector and 100 nM of p38 siRNA and/or PP5 siRNA or control siRNA. After that, FP was added for an additional 2 hours (top). Cells were lysed, and the luciferase activity was measured as described in Materials and Methods. The results are expressed as fold increase over basal. Data are representative of three separate experiments. (B) Bottom: ASM cells were transfected for 48 hours with 100 nM of p38 siRNA and/or PP5 siRNA or control siRNA. Cells were treated as above. Total mRNA was analyzed by real-time PCR. The values indicate expression of the steroid-inducible gene, GILZ, normalized to GAPDH mRNA levels and presented relative to the expression of untreated cells, which was set as 1. *P < 0.05 compared with untreated cells transfected with control siRNA. ^#P < 0.05 compared with FP-treated cells transfected with control siRNA. ^&P < 0.05 compared with FP-treated cells transfected with PP5 siRNA. (C) Cells were treated with FP (100 nM) for 2 hours and lysed, and whole cell lysate extracts were prepared and immunoprecipitated with anti-p38 or anti-PP5 antibodies and then assayed by immunoblot analysis. Results are representative of three separate blots. Data are representative of three separate experiments.