Figure 3. Ubiquitination activity of recombinant WT and mutant CHIP proteins.

In vitro ubiquitination activity assay was performed using WT and mutant CHIP as ubiquitin ligases and Hsc70 recombinant protein as substrate. Hsc70-ubiquitination (A and C) and self-ubiquitination activities (B and D) were explored on MBP-CHIP fusion proteins (A and B) and MBP-cleaved CHIP proteins (C and D). The reaction was incubated for 1 h at 37°C and samples were analyzed for both Hsc70- and self-ubiquitination by SDS/PAGE and immunoblotting using antibodies against Hsc70 and CHIP respectively. As a negative control, the WT CHIP protein was used in a separate reaction without adding ubiquitin.