A: km23-2 interacts preferentially with Smad3, relative to Smad2. 293T cells were transiently co-transfected with Smad2-Myc (lanes 1-4), Smad3-Myc (lanes 5-9), and RI-V5, RII-HA, or EV, together with or without km23-2-Flag as indicated. 28h after transfection, cells were treated as for Fig. 1A. Top panel, 293T cells were lysed, followed by IP/blot analyses using an anti-Myc Ab as the IP Ab and an anti-Flag Ab as the blotting Ab. Middle panel, the same membrane was blotted with an anti-Myc Ab to indicate expression of the IP’d Smads. Bottom panel, equal expression of transfected km23-2-Flag was verified by immunoblot analysis. The results shown are representative of two similar experiments. B: km23-1 interacts preferentially with Smad2, relative to Smad3. 293T cells were transiently co-transfected with Smad3-Myc (lanes 1-4), Smad2-Myc (lanes 5-9), and RI-V5, RII-HA, or EV, together with or without km23-1-Flag as indicated. Top panel, experiments were performed as in Fig. 6A. Bottom panels, equal expression of transfected Smad2-myc, Smad3-myc, and km23-1-Flag were verified by immunoblot analysis.