Response of Smad3 isoform to regulators of activin signaling and to TGFβ1 ligand. (A) Anti-phospho-Smad3 (top) and anti-Smad3 (bottom) immunoblots of LβT2 cells treated with vehicle (Con), activin A or activin B (30 ng/ml) in the absence or presence of follistatin (FST; 300 ng/ml) for 1 hour. Follistatin treatment abolished the induction of both full-length phospho-Smad3 and phospho-Smad3 isoform proteins by activin A and B. (B) Untreated (control) and activin A-treated LβT2 cells were transfected with empty vector. Additionally, LβT2 cells were transfected with expression vectors for TGFbRII and Alk5-HA, and treated with TGFβ1 (Tβ1) for 6 hours. Lysates were blotted with either anti-phospho-Smad3 antibody (left top), anti-Smad3 antibody (left bottom), or anti-HA (right top). Compared with activin A treatment (lane 2), TGFβ showed a much weaker induction of the phospho-Smad3 isoform.