RelA is required for transcriptional activation of Smad7 by TNF-α. (A) Northern blot analysis of Smad7 transcript levels in RelA+/+ and RelA−/− fibroblasts treated with TNF-α (10 ng/ml) or TGF-β (1 ng/ml) for the indicated time periods. The same blots were probed with GAPDH to control for RNA loading. (B) Smad7 mRNA in murine fibroblasts (NIH-3T3), Mv1Lu, and HSC. Cells were exposed to TNF-α for 1 hr. 18S rRNA was probed to control for loading. (C) RelA+/+ and RelA−/− fibroblasts were incubated with TNF-α (10 ng/ml), IL-1β (1 ng/ml), LPS (10 μg/ml), and IFN-γ (250 U/ml) for 1 hr, respectively. (D) RelA+/+ fibroblasts were incubated with TNF-α (10 ng/ml) for 1 hr in the absence or presence of various compounds including cycloheximide (10 μg/ml), SB203580 (10 μm), U0126 (10 μm), actinomycin D (10 μg/ml), or DMSO alone as control (28S rRNA as loading control).