TGF-β2 activation of c-Abl signaling and TGF-β2 activation of Smad signaling are independent. (A) Left: Smad3 KO MEFs (Smad3^–/–) were transfected with Flag-tagged c-Abl (c-Abl–Flag) and dominant negative Smad2 (myc-Smad2^D450E) as described in Methods. NIH-3T3 cells were treated as described in Figure 1. Following 30-minute stimulation in the absence (–) or presence (+) of 10 ng/ml TGF-β2, kinase activity of the transfected (c-Abl–Flag activity) or endogenous (c-Abl activity) c-Abl protein was determined using anti-Flag or K12 serum respectively. The corresponding total protein is indicated in the second and fourth panels. Right: NIH-3T3 or Smad2/3 KO cells were treated with (+) or without (–) TGF-β as described for the left panels. Following 30-minute stimulation, lysates containing equivalent protein were Western-blotted for the indicated proteins. (B) Left: NIH-3T3 cells were treated with (+) or without (–) TGF-β2 and/or imatinib as described in Figure 1. Following 30-minute stimulation, lysates containing equivalent protein were Western-blotted for phospho-Smad2 (p-Smad2), phospho-Smad3 (p-Smad3), or the corresponding total protein. Right: Abl^–/–Arg^–/– MEFs or Abl^–/–Arg^–/– fibroblasts stably expressing (+) WT-c-Abl or dominant negative c-Abl were treated with (+) or without (–) TGF-β2 for 30 minutes and processed for the indicated Smad protein as described for the left panels.