FIG. 2.

Effect of potassium depletion on TGF-βR internalization and/or Smad2 phosphorylation. (A) Internalization of ¹²⁵I-labeled GM-CSF through the chimeric TGF-βRs was performed in MB102-9 cells in the absence (▴) or presence (▪) of potassium. At the indicated times, the ratio of internalized to surface bound ligand was determined. The results represent the mean ± the SEM of three experiments done in duplicate. (B and C) At the top, serum-starved MB102-9 cells were left untreated (lanes 0) or were treated with 10 ng of GM-CSF/ml (B) (to activate chimeric TGF-βRs) or 10 ng of TGF-β/ml (C) (to activate endogenous TGF-βRs) for the indicated times. Immunoblotting with a phospho-specific Smad2 antibody was performed with equivalent protein lysates (100 μg) prepared from parallel plates treated with (+K) or without (−K) potassium as in panel A. The blot was stripped and probed with Smad2 antibody to confirm equal loading (lower half). In the bottom portions of panels B and C, the amount of Smad2-P relative to total Smad2 observed at the indicated time of ligand stimulation was quantified. The ratio obtained for the −K treatment was given the relative value of 1.0.