FIGURE 1.

Smad3 expression levels are down-regulated in EpRas cells and are regulated during the cell cycle. A, confluent EpH4 and EpRas cells were treated ± 2 ng/ml TGF-β1 for 45 min. Whole cell extracts were prepared from confluent cells, and equal amounts of protein were analyzed by Western blotting using antibodies against the Smad2/3 and 4 and Grb2 as a loading control. B, Smad3 protein expression is regulated during the cell cycle. Synchronized, quiescent EpH4 and EpRas cells were prepared by contact inhibition for 72 h. Actively cycling, low confluency EpH4 and EpRas cells were prepared by synchronizing the cells by contact inhibition and then plating into fresh medium for 24 h. Cells were either unstimulated or treated with 2 ng/ml TGF-β1 for 1 h as indicated. Whole cell extracts were prepared, and equal amounts of protein were analyzed by Western blotting using antibodies against Smad2/3 and Smad4 and Grb2 as a loading control. C, cycling EpH4 and EpRas cells were treated with 2 ng/ml TGF-β for the times indicated. Whole cell extracts were blotted with antibodies against phosphorylated Smad2, phosphorylated Smad3, Smad2/3, and MCM7 as a loading control. D, EpH4 and EpRas cells were synchronized by contact inhibition and then plated into fresh medium for the number of hours indicated. Cells were harvested and analyzed by FACS, to determine the number of cells in G₁ (black bar), S (white bar), or G₂/M (gray bar) and by Western blotting, using antibodies against Smad3 and Grb2 as a loading control.