FIGURE 3.

mDia2 is polyubiquitinated. A, HEK293T cells were transfected with Myc-mDia2, HA-ubiquitin, or co-transfected with both plasmids. Lysates were immunoprecipitated (IP) for either Myc or HA. Immunoblots (IB) of lysates and immunoprecipitations were probed with anti-Myc or anti-HA to examine the extent of ubiquitination. B, HeLa cells were incubated with 10 μm MG132 for 18 h. Lysates were immunoprecipitated using anti-β-catenin or anti-mDia2. Immunoblots were probed with anti-ubiquitin, anti-β-catenin, or anti-mDia2 (1358) to examine endogenous ubiquitination. C, HEK293T cells were co-transfected with Myc-mDia2 and HA-ubiquitin. Lysates were immunoprecipitated with anti-Myc. HeLa cells were incubated with 20 μm MG132 for 4 h. Lysates were immunoprecipitated with anti-β-catenin. Immunoprecipitations were immunoblotted with anti-Myc and an antibody specific to polyubiquitin chains.