Figure 3

Involvement of ERK1/2 in TGF-β1-induced MMP-9 expression in RBA-1 cells. (A) Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of U0126. (B) Cells were pretreated with U0126 (10 mM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (C) Time dependence of TGF-β1-stimulated ERK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated time intevals. (D) Cells were treated with TGF-β1 (15 ng/ml) for 10 min in the absence or presence of U0126 (10 μM). The whole cell lysates (C, D) were subjected to 10% SDS-PAGE and analyzed using an anti-phospho-ERK1/2 or anti-GAPDH (as an internal control) antibody. (E) Cells were transfected with an empty vector (pcDNA3, as a control), a dominant negative mutant of ERK1 (ΔERK1) or ERK2 (ΔERK2) for 24 h, and then exposed to TGF-β1 for 16 h. The conditioned media (A, E) were analyzed gelatin zymorgraphy. Data are expressed as mean ± SEM (C) or mean (A, B, D, E) of at least three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to vehicle (C) or TGF-β1 (A, B, D, E) alone. The figure represents one of three individual experiments.