Figure 3

BK induces MMP-9 expression via p47^phox translocation in RBA-1 cells. (A) Cells were pretreated without or with Apocynin (Apo, 10 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. (B) Cells were pretreated with or without Apo (10 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. (C) Cells were pretreated without or with Apo (10 μM) for 1 h treated with 10 nM BK for the indicated time intervals or 3 min. The membrane and cytosol fractions were prepared and analyzed by Western blotting. The p47^phox translocation was also confirmed by immunofluorescent staining. (D) Cells were pretreated without or with Apo (10 μM) for 1 h before exposure to 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. (E) Cells were transfected with scramble (scra) or p47^phox siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected for zymography of MMP-9 or Western blotting to determine the levels of Nox2 and GAPDH (as an internal control). Data are expressed as the mean ± SEM (n = 3). ^*P < 0.05; ^#P < 0.01, as compared with the respective values of cells stimulated with vehicle (C) and BK (A, B, D, E) only. The figure represents one of three similar experiments.