Fig. 7. XMAN1 preferentially associates with BMP-responsive Smads. (A) XMAN1 associates with XSmad1 in vivo. Myc-tagged wild-type (MT-XMAN1), C-terminal (MT-CT) and N-terminal (MT-CT) constructs of XMAN1 were co-expressed with HA-tagged XSmad1 in Xenopus embryos as indicated. Extracts prepared from stage 10.5 embryos were immunoprecipitated with an anti-HA antibody. The precipitates were analyzed by western blotting with an anti-Myc antibody (upper panel). Middle and lower panels show the expression levels of XMAN1 and XSmad1 proteins, respectively. (B) XMAN1 associates with endogenous XSmad1. Extracts prepared from stage 10.5 embryos injected with or without MT-CT were immunoprecipitated with anti-Smad1 antibodies, and the precipitates were immunoblotted with anti-Myc antibody (upper panel). Middle and lower panels show the expression levels of MT-CT and endogenous XSmad1 proteins, respectively. (C) XMAN1 interacts preferentially with BMP-responsive Smads in vitro by GST pull-down assays. [³⁵S]Met-labeled in vitro translated Smad and GFP products were incubated with purified GST or GST-XMAN1-CT and subjected to SDS-PAGE. % binding, percentage of the intensity of each band in the lane of GST-CT against the corresponding total input.