(A) Following an initial 24 hr culture period with medium containing 50 μg/mL ascorbic acid, USCs were treated with or without 5 ng/mL TGF-β for 4 hours. Total cellular proteins were harvested and immunoprecipitations were performed using pSmad3 antibody. Immunoprecipitated proteins were run out on polyacrylamide gels, transferred to nylon membranes and Western blotting was performed to detect HDAC4 and HDAC5. (B) Following initial culturing in medium containing 50 μg/mL ascorbic acid, USCs were infected with pRetro-shSki or a nonsense shRNA virus. Twenty four hours later, cells were treated with or without 5 ng/mL TGF-βfor 4 hours. Total cellular protein was harvested and immunoprecipitations were performed as described in Experimental Procedures using pSmad3 antibody. Immunoprecipitated proteins were run out on polyacrylamide gels, transferred to nylon membranes and Western blotting was performed to detect HDAC4 and HDAC5. Blots shown in panels (A) and (B) are representative of results obtained from experiments that were repeated 3 times.