Figure 5

NF-κB (p65) is essential for ET1-1-induced COX-2 expression. (A, B) Cells were treated with 10 nM ET-1 for 6 h in the absence or presence of Bay11-7082. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR as described in Figure 1. (C) Time dependence of ET-1-stimulated p65 NF-κB translocation by subcellular isolation, Western blot, and immunofluorescent stain. (D) Cells were pretreated with U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), BQ-788 (1 μM), or Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. The nuclear fraction was analyzed by Western blot. (E) Cells were transfected with p65 siRNA and then exposed to ET-1 for 6 h. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-p65, anti-Lamin A, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; ^#P<0.01 as compared with ET-1 alone).