Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4 null fibroblasts.

<p>(<b>A</b>) EF7WT (wild-type) and EF7KO (Smad4 deficient) fibroblasts were transduced with recombinant adenovirus for HA-tagged Smad4 (RAdSmad4), or with empty control virus RAd66 at MOI 100 (EF7WT) or 300 (EF7KO). After 36 h incubation cell lysates were harvested and analyzed by Western blotting to detect the levels of endogenous and exogenous Smad4. Anti-HA antibody was used to detect adenovirally delivered Smad4 (upper panel) and anti-Smad4 to detect endogenous Smad4 (lower panel). (<b>B</b>) EF7KO fibroblasts were infected with adenoviruses RAdSmad4 or RAd66. After 36 h incubation the cells were stimulated with TGF-β1 (5 ng/ml) for different periods of time, as indicated. Total RNA was extracted and analyzed by qRT-PCR to determine TIMP-3 and PAI-1 mRNA levels. mRNA expression (mean ± SEM from two separate experiments, both run with duplicates) is shown relative to 18S ribosomal RNA. *p<0.05, t-test).