FIG. 3.

Smad2 and Smad3 nuclear translocation requires receptor internalization. (A) MB102-9 cells were left untreated (lanes 0) or were treated with 10 ng of GM-CSF/ml (lanes G) or 10 ng of TGF-β/ml (lanes B) with (+K) or without (−K) potassium at 37°C to activate chimeric or endogenous TGF-βRs, respectively. Nuclear fractions were analyzed by immunoblotting with a Smad2 (top) and Smad3 (bottom) antibody. (B) Cells with (i, iii, and v) or without (ii, iv, and vi) potassium were left untreated (i and ii), treated with 10 ng of GM-CSF/ml (iii and iv) to examine the response to chimeric TGF-βR activation, or treated with 10 ng of TGF-β/ml (v and vi) to examine the response to endogenous TGF-βR activation for 20 min at 37°C. Smad3 was visualized by immunofluorescence as described in Materials and Methods.