Figure 5.

Interaction between p47^phox and Nox contributes to MVS-induced HO-1 expression. (A) HPAEpiCs were transfected with p47^phox siRNA and then incubated with vehicle or MVS (30 μM) for the indicated time intervals. The levels of p47^phox phosphorylation, total p47^phox, and GAPDH were determined by Western blot using anti-phospho-p47^phox, anti-p47^phox, or anti-GAPDH antibody. (B) Cells were pretreated with MVS (30 μM) for the indicated time intervals. The cytosol and membrane fractions were prepared and analyzed by Western blot using anti-p47^phox, anti-GAPDH, or anti-Gαs antibody. HPAEpiCs were treated with MVS for the indicated time intervals. The levels of (C,D) NADPH oxidase activity and (E–G) ROS generation (H₂O₂ or O₂^.–) were determined by ELISA or immunofluorescence (IF), reaching a maximal response within 15 min for Nox activity or 30 min for ROS. MVS stimulates Nox activity leading to ROS production, cells were pretreated with Nox or ROS inhibitors for 1 h, and then incubated with vehicle or MVS (30 μM) for (D) 15 min for Nox activity or (F,G) 30 min for ROS generation. MVS-induced Nox activity and ROS production were reduced by pretreatment with NAC (1 mM), APO (1 mM), and DPI (3 μM) in HPAEpiCs. Data are expressed as mean ± SEM from five independent experiments (n = 5). ^#P < 0.01 compared with the respective significantly different values as indicated.