Figure 1.

Runx2 interacts with CHIP. (A) Runx2 physically interacts with CHIP in vitro. A GST pull-down assay was performed with the purified GST or GST-CHIP protein and Myc-Runx2 protein expressed by 293T cells. (B) Interaction of Runx2 with CHIP occurs in the nucleus of mammalian cells. Cytoplasmic (Cy) and nuclear (Nu) extracts were used in coimmunoprecipitation assays with anti-HA antibody, and the precipitated complexes were analyzed with an anti-Myc antibody against Myc-Runx2. (C) Endogenous Runx2 and CHIP proteins interact. MC3T3-E1 cells were treated with 10 μM MG132 for 4 h before lysis. (top) Whole cell lysates were immunoprecipitated with an anti-CHIP antiserum or a preimmune serum (Pre). The immunoprecipitates were visualized with an anti-Runx2 antibody (65 kD*; Santa Cruz Biotechnology, Inc.). (bottom) Reversed coimmunoprecipitation assays were performed using anti-Runx2 antibodies (MBL International) and IgG. (D) The C terminus of Runx2 interacts with CHIP. Schematic illustration of Runx2 and its truncated constructs is shown on top. QA, QA domain; RHD, runt homology domain; NMTS, nuclear matrix targeting signal. A GST pull-down assay was performed using the purified GST-CHIP protein and different deletions of the hRunx2 protein expressed in mammalian cells. (E) TPR domain of CHIP is critical for the interaction with Runx2. Three main functional domains of CHIP (TPR, tetratricopeptide repeats; U-box, U-box domain; Charged, charged domain) are schematically illustrated on top. A GST pull-down assay was performed using GST-fused CHIP, and the deletion proteins to pull down the mammalian cell expressed Myc-Runx2 protein. (F) Mutation CHIP(K31A) impairs the ability of CHIP to interact with Runx2. Lysates were prepared from 293T cells expressing HA-tagged CHIP, CHIP(K31A), or CHIP(H261Q) together with the indicated Myc-Runx2. Coimmunoprecipitation and immunoblotting were performed as described in A. The result from a longer exposure (5×) for the immunoblot is also presented (top).