WWOX binds to SMAD3 and relocalizes it to the cytoplasm. (A) Co-immunoprecipitation of endogenous WWOX and SMAD3 from MCF10 cells. Whole cell lysates were immunoprecipitated with either rabbit IgG (control), anti-WWOX, anti-SMAD3 or anti-RUNX2 antibodies. The immunoprecipitates were immunoblotted with anti-WWOX and anti-SMAD3 antibodies as indicated. RUNX2 co-IP was used as a positive control for both WWOX and SMAD3 interactions. (B) MCF10 cells were transfected with Flag-SMAD3 and pulldowns were performed with the indicated GST fusion proteins either wild-type WW1 and 2 domains (GST-WT) or mutant WW1 (W44F/P47A) (GST-mut1) or GST alone. Bound protein was detected by SMAD3 immunoblot. (C) Confocal microscopy of MCF10 cells transfected with pcDNA-Myc-WWOX and treated with TGFβ1. Note how SMAD3 (green) localizes to the nucleus in cells with no Myc-WWOX expression (red) but undergoes intracellular redistribution mostly to the cytoplasmic compartment in cells with ectopic WWOX expression (red). Images taken using a Zeiss LSM510 META confocal microscope and the 100X objective.