Fig. 6. Conjugation of Pub2p with ubiquitin. (A) A schematic representation of the fusion constructs. Ubiquitin (Ub) was fused to the C-terminus of glutathione-S-transferase (GST). A Myc epitope tag was fused to the C-terminus of the HECT domain of wild-type Pub2p (HECT^Pub2-Myc) or to that of Cys639 Ala mutant HECT domain (HECT CA^Pub2:Myc). (B) Association of the Pub2 HECT domain with ubiquitin. GST-Ub and HECT-Myc fusion proteins were co-expressed in wild-type cells (MM72-1D), which were incubated in thiamine-free EMM2 for 20 hours. GST-fusion protein was pulled down from crude cell-free extracts by glutathione beads. Pulled-down proteins were resolved by SDS-PAGE on 10% gels after treatment (+) or mock treatment (-) with 100 mM DTT. Western blots were probed with an anti-Myc antibody (9E10).