Fig. 4. Endofin modulates Smad1 phosphorylation and nuclear translocation. (A) Silencing of endofin expression by RNA interference inhibited Smad1 phosphorylation. C2C12 cells were transfected with Flag-tagged Smad1 and either vector, construct for GFP or endofin siRNA. On the third day following 3-hour BMP2 treatment, Flag-tagged Smad1 was precipitated with anti-flag and its phosphorylation level was detected with anti-phospho-Smad1. (B) Endofin, but not SARA, regulates BMP-specific phosphorylation and intracellular translocation of Smad1. C2C12 cells were transfected with either HA-SARA or HA-Endofin, then stimulated with BMP (100 ng/ml) for 3 hours. After fixation with paraformaldehyde (PFA), cells were immunostained with either anti-HA (green) or anti-phospho-Smad1 (red). Nuclei were visualized using Hoechst 33342. Representative images are shown. Bar, 20 µm.