FIG. 2.

Immunoprecipitation assays showing CHIP-Smad interaction in vivo. (A) Overexpressed Flag-tagged Smad1 precipitated Myc-tagged CHIP in the presence of MG132. Myc-tagged CHIP was cooverexpressed with Flag-tagged Smad1 in the absence or presence of 50 μM MG132. The precipitated band was as indicated. (B) CHIP interacts with Smad1 and Smad4 in mammalian cells. 293T cells were transfected with plasmids expressing Flag-tagged full-size Smad1 or Smad4 or HA-tagged full-length CHIP as indicated. The whole-cell lysates were used in immunoprecipitation (IP) with anti-Flag M2 antibody and blotted with anti-HA antibody (IB) (upper panel). The levels of F-Smads in the whole-cell lysates (middle panel) and HA-CHIP (bottom panel) have been shown as indicated. MG132 was added to the transfected cells. (C) Myc-CHIP interacts with endogenous Smad1 in vivo. HepG2 cells were transfected with Myc-tagged CHIP or a constitutively active form of BMPRIB(QD). After 24 h of transfection, cells were metabolically labeled with ³⁵S-methionine and IP was then carried out. SDS-PAGE gel was dried and visualized by autoradiograph. The expression of Myc-CHIP and endogenous Smad1 are shown in the middle and lower panels separately. (D) EndogenousCHIP interacts with endogenous Smad1 in vivo. The HepG2 cells were metabolically labeled with [³⁵S]methionine. The cell lysate was immunoprecipitated with either anti-Smad1 antibody or anti-CHIP serum. The precipitated complex with anti-Smad1 antibody was then reimmunoprecipitated with either anti-Smad1 antibody to show the expression of endogenous Smad1 (middle panel) or anti-CHIP serum to show the interaction of Smad1 and CHIP (top panel). The precipitated complex with anti-CHIP serum was reimmunoprecipitated with anti-CHIP serum to show the endogenous expression of CHIP (bottom panel) in the cells. Mouse IgG and preimmune rabbit serum (Pre-S) were always used as negative controls for the reimmunoprecipitation as indicated.