FIG. 5.

CHIP as an E3 ligase targets myocardin for proteasomal degradation. (A) 293 cells were transfected with a Flag-myocardin, Myc-CHIP, or vector plasmid and treated with cycloheximide (CHX, 10 μg/ml) at the indicated time points. The half-life of myocardin was determined by immunoblotting with anti-Flag antibody and quantified in the presence of a CHIP or vector plasmid (n = 3, means ± SEM). (B) The effect of CHIP on steady-state levels of myocardin was examined by transfection with a Myc-CHIP or vector plasmid in the presence of MG132 for 4 h. (C) The ubiquitination of endogenous myocardin in SMCs was tested by transfection with a Myc-CHIP or vector plasmid. Cell extracts were immunoprecipitated (IP) with antimyocardin antibody and immunoblotted (IB) with an anti-Ub (top) or antimyocardin (middle) antibody. The expression of myocardin and CHIP in the cell extracts was determined with antimyocardin or anti-Myc antibody (bottom). (D) 293 cells were transfected with a His-Ub, Myc-CHIP, or CHIP ΔU-box mutant plasmid in the presence of Flag-myocardin WT or mutant protein 1-713 and incubated with MG132 for 4 h before harvesting. Ub-conjugated proteins were purified by nickel chromatography and analyzed by immunoblotting with anti-Flag (top) or anti-His (middle) antibody. The expression of myocardin and CHIP in the cell extracts was determined with anti-Flag or anti-Myc antibody (bottom). (E) In vitro ubiquitylation reactions were performed with purified Ub, E1, E2-Ubc5b, GST alone, GST-CHIP, and GST-CHIP ΔU-box mutant protein in the presence of Flag-myocardin WT or 1-713 mutant protein. Reaction products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-Ub (top) or anti-Flag antibody (bottom).