Figure 4

A) AMSH-2 interacts with Smad2 and Smad7, but not with Smad3 or Smad4. Vector or AMSH-2 V5 construct were coexpressed in 293T cells together with the Smad proteins as indicated on the top of the figure. Lysates were immunoprecipitated with anti-V5 antibody and western blotted with anti-Flag or anti-HA antibodies. To control for Smad and AMSH-2 protein expression part of the lysates were immunoprecipitated with anti-Flag, anti-HA or anti-V5 antibodies and western blotted with the same antibodies. B) Smad7 inhibitory effect on TGF-β signaling pathway is rescued by AMSH-2. HepG2 cells were cotransfected with 3TP-lux reporter together with the empty vector or 10 ng/ml of Smad7 with or without 0.3 μg/ml of AMSH-2 Myc construct. Twenty-four hours posttransfection, cells were serum-starved and left untreated (white bars) or stimulated (black bars) with 5 ng/ml TGF-β1 for 16–20 h before luciferase and β-galactosidase activities were measured.