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. 2003 Dec;23(24):9081–9093. doi: 10.1128/MCB.23.24.9081-9093.2003

FIG. 4.

FIG. 4.

Smad6 interacts with the transcription corepressor CtBP in vivo. (A) Smad6 coimmunoprecipitates with CtBP. 293T cells were transfected with HA-tagged Smad6 and myc-tagged CtBP. Cell lysates were immunoprecipitated (IP) with anti-HA antibodies (12CA5 or HA1.1)and then immunoblotted (IB) with an anti-Myc polyclonal antibody to detect Smad6-bound CtBP (upper panel) and anti-HA to determine the level of immunoprecipitated Smad6 (bottom). Whole-cell lysates (WCL) were also directly immunoblotted with anti-Myc or anti-HA antibodies to demonstrate the expression of transfected CtBP and Smad6 (lanes 7 and 8). (B) Smad6 interaction with CtBP is specific. Immunoprecipitation-Western analysis was conducted as for panel A, except that anti-Flag and anti-HA antibodies were used for IP and IB, respectively. (C) Endogenous interaction between Smad6 and CtBP. The IP-Western blot procedure was carried out similarly to that in panel A, with the antibodies indicated. (D) Smad6 is colocalized with CtBP. Immunofluorescence was performed as in Fig. 2A.