Fig. 7. Xmsx-1 physically interacts with Smads. (A) Xmsx-1/Xsmads complexes were detected by immunoprecipitation. 293T cells were transiently transfected with Flag-tagged Xmsx-1 alone (lane 2) or the indicated HA-tagged Xsmads alone (lanes 3-6), or cotransfected with Flag-tagged Xmsx-1 and each HA-tagged Xsmad (lanes 7-10). Lysates were assayed either by immunoprecipitation with an anti-Flag antibody followed by western blotting with an anti-HA antibody (upper panel) or by western blotting the whole lysate with an anti-HA antibody (lower panel). Lane 1 is a nontransfected control. (B) Xmsx-1 additively participates in the Xsmad2/Xsmad4ß complex. 293T cells were transiently cotransfected with glutathione S-transferase (GST)-tagged Xsmad4ß and HA-tagged Xsmad2 together with increasing amounts of HA-tagged Xmsx-1 (lanes 3-6). Lysates were assayed either by GST pull-down analysis followed by western blotting with an anti-HA antibody (upper panel) or by western blotting the whole lysate with an anti-HA antibody (lower panel). As negative controls, GSTvector expressing GST protein alone was cotransfected either with HA-tagged Xsmad2 (lane 1) or with HA-tagged Xmsx-1 (lane 2). (C) Xmsx-1 excludes xFAST-1 from Xsmad2/4 complex in a dose-dependent manner. Cells were cotransfected with the indicated amount of GST-xFAST-1, HA-Xsmad2, Xsmad4ß and HA-Xmsx-1 constructs. Lysates were assayed as in B.
