Fig. 2.

EGF does not decrease TGF-β-induced nuclear accumulation of Smad2/3 in RFL-6 fibroblasts. A: postconfluent serum-starved cells were treated for 1 h with or without 10 ng/ml EGF, 2 ng/ml TGF-β1, or EGF + TGF-β1. Cytosolic and nuclear extracts, as well as whole cell lysates, were prepared, and 30 μg of total protein from each sample were analyzed by Western blot with antibodies against Smad2/3 and β-actin. β-Actin was probed as a loading control. Results are representative of an experiment that was repeated 3 times. B: cells were grown on coverslips and to 50% confluence, serum-starved for 4 h, and then treated for 1 h with or without 10 ng/ml EGF, 2 ng/ml TGF-β1, or EGF + TGF-β1. Cells were fixed and stained with anti-Smad2/3 primary and Cy3-conjugated secondary antibodies, and their phase and fluorescent images were obtained (original magnification ×250). Results are representative of an experiment that was repeated 3 times.