For immunoprecipitation, cells were lysed using the abovedescribed lysis buffer with freshly added 20 mM iodoacetamide. A total of 500 mg of cell lysates were incubated with a monoclonal anti-ubiquitin antibody (1:100) at 4uC overnight, followed by 2 h incubation with protein G-agorose beads (Amersham). Immunocomplexes were washed five times with the lysis buffer containing 20 mM iodoacetamide, and then boiled for 5 min in the 26 nonreducing sample buffer which lacks both b-mercaptoethanol and DTT, but with addition of 20 mM iodoacetamide. After centrifugation, the precipitated proteins were separated by SDS-PAGE. Ubiquitin conjugates were analyzed by immunoblot using polyclonal anti-3D pol antibody.