Fig. 7. SRF inhibits Activin-induced formation of FAST-1-Smad2 complex. (A) Stable Mv1Lu cells expressing exogenous SRF and control cells were incubated in the presence or absence of 25 ng/ml Activin A for 1 hour. Smad2 phosphorylation was analyzed by immunoblotting with antiphospho-Smad2 antibody. Expression of Smad2 and SRF was monitored by immunoblotting with anti-Smad2 and anti-HA antibodies. (B) Stable Mv1Lu cells expressing exogenous SRF and control cells were treated as in A. Cell lysates were immunoprecipitated with anti-Smad2 antibody and then immunoblotted with anti-Smad4 antibody. Expression of Smads and SRF was monitored by immunoblotting with anti-Smad2, anti-Smad4 and anti-HA antibodies. (C) 293T cells were co-transfected with the indicated constructs and harvested 36 hours after transfection. Cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti-Myc antibody. Expression of Flag-Smad2, Myc-FAST-1, HA-ALK4^* and SRF was monitored as indicated. (D-G) Rescue by FAST-1 mutants of the axial defects caused by gain- or loss-of-function of XSRF. Four-cell stage embryos were injected dorsally with a combination of the indicated reagents (2 ng wt XSRF; 20 pg FAST-VP16^A; 60 ng XSRF MO; 2 pg FAST-En^R) and cultured to stage 31.