In Vitro Ubiquitination Assay

An in vitro ubiquitination assay was performed as previously described (42). Briefly, ubiquitination reaction mixtures were prepared first by combining 0.125 μM E1 (Ube1) and 1 μM E2 (UbcH5b) (Boston Biochem), 200 μM ubiquitin (Sigma), and the appropriate volume of 10× reaction buffer (50 mM HEPES, pH 7.0, 50 mM NaCl, 20 mM ATP, 40 mM MgCl₂), followed by a 30-minute incubation at 37°C. In parallel, a total of 3 μM of purified CHIP was combined on ice with Hsc70 substrate recognition domain (GST-Hsc70395-646) in 50 mM HEPES (pH 7.0) and 50 mM NaCl. Control reactions were also set up in the absence of ATP. After addition of the two mixtures, the reactions were incubated for 1 hour at 20°C and then stopped by the addition of SDS-PAGE sample buffer supplemented with 50 mM EDTA. The quenched reactions were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with either anti-GST HRP-conjugated antibody (Abcam) or anti-ubiquitin (catalog no. SC: 8017; Santa Cruz Biotechnology), which was detected with horse anti-mouse-HRP antibody (Cell Signaling). The concentrations provided for all purified proteins used in the reactions reflect their final reaction concentrations.