Figure 5.

CHIP interactions with Ube2e2 and UbcH7. A. Autoubiquitination of CHIP in the presence of Ube2e2 catalytic domain (Ube2e2-cat) probed by western blotting. Anti-ubiquitin staining confirms the formation of polyubiquitin chains. Anti-CHIP staining shows that a fraction of CHIP is modified by mono- and polyubiquitin, migrating as higher-molecular weight bands. B. In vitro ubiquitination measurements of CHIP with Ube2e2 and UbcH7 carried out using the ELISA-based assay. His-CHIP bound to Nickel-NTA coated microplates was autoubiquitinated with FLAG-Ubiquitin in the presence of E2 enzymes as shown. UbcH5b was used as the positive control; a reaction containing UbcH5b but no His-CHIP was used as a negative control ("-CHIP"). Counts were normalized by the value measured for the positive control.