Figure 5.

CORM-3-induced HO-1 expression is mediated through p42/p44 mitogen-activated protein kinase (MAPK). (A) RBA-1 cells were pretreated with various concentrations of U0126 for 1 h, and then incubated with 30 μM CORM-3 for 6 h. The levels of HO-1 and GAPDH (as an internal control) protein expression were determined by Western blot. (B) RBA-1 cells were pretreated with 10 μM U0126 for 1 h and then incubated with 30 μM CORM-3 for 4 h. The levels of HO-1 mRNA were determined by real-time PCR (Open bars). Cells were transiently transfected with HO-1 report gene together a β-galactosidase plasmid, pretreated with U0126 (10 μM) for 1 h, and then incubated with CORM-3 for 1 h. Promoter activity was determined in the cell lysates (Gray bars). (C) RBA-1 cells were transfected with p44 or p42 siRNA and then incubated with 30 μM CORM-3 for 6 h. The levels of total p42, p44 and HO-1 were determined by Western blot. (D) RBA-1 cells were pretreated with 10 μM U0126, 10 μM SU6656, 10 μM PF431396, or 3 μM Gö6983, for 1 h and then incubated with 30 μM CORM-3 for the indicated time intervals. Phosphorylation of p42/p44 MAPK was determined by Western blot using an anti-phospho-p42/p44 MAPK or anti-p42/p44 MAPK antibody. Data are expressed as the mean ± SEM of three independent experiments (n = 3). ^#p < 0.05, as compared with the cells exposed to CORM-3 alone.