Fig. 7. Modulation of SMAD4 expression by eIF2S2. (A) SiHa cells were transfected with p3x-Flag-CMV10-eIF2S2 or Flag-Smad4 or their combination, and the Smad4 and Flag expressions were detected by immunoblot analysis. (B) SiHa cells were transiently transfected with eIF2S2 siRNA (10 nM) or control siRNA (10 nM) and cell lysates were separated by SDS-PAGE and immunoblotted with Smad4 and eIF2S2. GAPDH expression was used as the loading control. (C) Protein extracts from clones of NIH3T3 cells stably transfected with vector alone or p3x-Flag-CMV10-eIF2S2 were subjected to Western blot analysis to detect Smad4 expression. Flag and eIF2S2 were detected to confirm transfection efficiency. (D) SiHa cells were incubated with the proteasomal inhibitor MG132 before the transfection of cells with p3x-Flag-CMV10-eIF2S2 or empty vector. Smad4 expression was detected by Western blot analysis. All experiments were performed in triplicate.
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