TGFβ-induced nuclear accumulation of Smad2 and Smad3 in EpH4 and EpRas cells. (A) COS cells transfected with Flag-tagged versions of Smad1, Smad2, Smad3, or Smad4 were subjected to Western immunoblotting with anti-Flag antibodies as a positive control, or antibodies raised against the linker region of Smad2. (B) Untransfected EpH4, EpRas, and Mv1Lu cell extracts were subjected to Western immunoblotting with affinity-purified anti-Smad2/Smad3 (top) or nonimmune serum (bottom). (Asterisks) Nonspecific bands. (C) EpRas and EpH4 cells were stimulated with TGFβ for 30 min. Cell lysates were subjected to Western immunoblotting with either antibodies against receptor-phosphorylated Smad2 (top) or anti-Smad2/Smad3 antibodies (bottom). (D) In a similar experiment, endogenous Smad2 and Smad3 were visualized by anti-Smad2/Smad3 immunofluorescence. (E) EpH4 (█) and EpRas (▴) cells were treated with TGFβ for different time periods. Following immunofluorescence staining, the percentage of cells with Smad2/Smad3 staining predominantly or exclusively in the nucleus was determined.