Fig. 5. Interaction of eIF2S2 and SMAD4 in SiHA and HeLA. (A) Immunoprecipitation in SiHa was performed with anti-SMAD4 monoclonal antibody (S4) or mouse control IgG. Whole lysate (WCL), supernatant (Sup), and immunoprecipitate (IP sample) were separated by SDS-PAGE and immunoblotted with either eIF2S2 or SMAD4 antibody. (B) HeLa cells were co-transfected with plasmids that harbor full-length eIF2S2, N-terminal-eIF2S2 (aa 1-140), or C-terminal-eIF2S2 (aa 141-333) in presence of full-length SMAD4 for BiFC assay (left panel). Full-length SMAD4 co-transfected with SMAD2-MH1 and -MHC2 was used as the negative and positive control, respectively. The N-terminal domain of eIF2S2 (aa 1-140) was co-transfected with plasmids that harbor each SMAD4 domain, and their interaction was visualized by BiFC assay (right panel). All experiments were performed in triplicate.
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