Fig. 1. Endofin interacts with Smad1. (A) Co-immunoprecipitation (co-IP) of endofin with Smads. Flag-tagged Smad1 or Smad2 was co-transfected with HA-tagged endofin into COS1 cells. The lysate was subjected to co-IP with anti-HA antibody and probed with anti-Flag antibody, or co-IP with anti-Flag and probed with anti-HA. (B) Confirmation of the interaction between Smad1 and endofin by protein-fragment complementation assays (PCA). The complementary fragments (YFP1 and YFP2) of YFP were fused with Smad1 and endofin, respectively, to create YFP1-Smad1 (a.a. 1-158 of YFP) and endofin-YFP2 (a.a. 159-239 of YFP), and then transfected into COS1 cells. The interaction between endofin and Smad1 is indicated here as yellow fluorescence. Transfection of pcDNA3, co-transfection of pcDNA3-YFP1 and pcDNA3-YFP2 and co-transfection of YFP1-Smad1 and Hoxa1-YFP2 serve as negative and positive controls, respectively (Li et al., 2006). Bar, 10 µm. (C) Colocalization of Smad1 and endofin. Upper panel: C2C12 cells were transfected with HA-Endofin and Flag-Smad1. After transfection for 36 hours, cells were processed for immunofluorescence using antibodies against HA (rabbit polyclonal) and Flag (mouse monoclonal). Lower panel: Endogenous protein co-localization. Naive C2C12 cells were subjected to immunofluorescent microscopy using rabbit polyclonal anti-endofin and mouse monoclonal anti-Smad1. Nuclei were counterstained with Hoechst 33342. Representative images are shown. (D) Endofin interacts with both Smad1 and PP1c. COS1 cells were transfected with Flag-Smad1, HA-tagged endofin and Myc-tagged PP1c. Cells were treated with rhBMP2 (200 ng/ml) for 2 hours before lysis. The lysate was subject to co-immunoprecipitation with antibody against HA, the presence of Smad1 and PP1c in the precipitate was detected with anti-Flag and anti-Myc antibodies, respectively. (E) Co-IP of endogenous endofin, Smad1 and PP1c. C2C12 cells were cultured until subconfluent, with or without treatment of rhBMP2 (200 ng/ml, 2 hours), cells were lysed, and the lysate was subjected to immunoprecipitation with antibody against either endofin or SARA. The presence of Smad1 and PP1c in the precipitate was detected by anti-Smad1 and anti-PP1c antibodies. Bar, 5 µm.