The effect of inhibition of MAPK on phosphorylation and nuclear translocation of Smad2 induced by TGF-β1.

(A) Phosphorylation of Smad2 was not affected by treatment with 10 μM PD98059 for 2 hours followed by addition of 10 ng/mL TGF-β1 for an additional 24 hours. (B) There was no significant difference observed in the percentage of nuclei positive for Smad2 immunostaining (of 1000 cells counted in 5 different fields) 18 hours after treatment with 10 ng/mL TGF-β1 and 10 nM ATRA with or without the addition of 10 μM PD98059 (10 μM PD) at 2 hours prior to the other treatments (means ± SEMs, n = 3). (C) The p44/42 MAP kinase activity is activated independently of the particular pathway of differentiation. Proteins were immunoprecipitated with anti–phospho-p44/42 MAP kinase antibody as described in “Materials and methods,” and the immunoprecipitates were subjected to an in vitro kinase assay with the use of the Elk-1 fusion protein. Reaction mixtures were separated by SDS-PAGE and immunoblotted with anti–phospho–Elk-1 antibody. The data shown are representative of 3 experiments with similar results.