TGF-β signalization pathway is amplified and complex formation is modified in the absence of Gfi-1B. (A) Increase of Smad2 phosphorylation in the absence of Gfi-1B. shControl- or shGfi-1B–transduced K562 cells were stimulated or not with TGFβ1 (R&D Systems) for 1 hour. Cell lysates were prepared and analyzed by Western blotting with antibodies indicated on the left of the figure (as controls, Smad2/3 and β-actin). Three experiments with different samples were performed. (B) Complex formation with activated Smad2. Proteins from shControl- or shGfi-1B–transduced K562 cells were immunoprecipitated (IP) with an antibody against Smad2/3 (or IgG as control) and immunoblotted (IB) with antibodies recognizing Smad2/3, P-Smad2, Smad4, and TIF1-γ. Inputs are shown. These results are representative of 3 experiments with different samples.