FIG. 1.

In vitro ubiquitin ligase activity of p28 and M143R. Purified GST fusion proteins were combined in an in vitro ubiquitination reaction with E1, UbcH5a (E2), ATP, and ubiquitin. Reactions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were probed with anti-ubiquitin (P4D1; Santa Cruz Biotechnology). Western blotting with anti-ubiquitin shows the appearance of high-molecular-mass ubiquitin adducts in reactions containing either GST-p28, GST-M143R, or GST-ICP0 compared to reactions with no GST-fusion, GST alone, the C173S/C176S double mutants of both p28 and M143R, or p28(1-184).