Fig. 3. C91 in UBC domain is required for in vitro E2 activity of UBE2W. (A) Sequence alignment of UBE2W homologues. The col-ored and shaded region represents con-served residues. C91, marked with bold-face in the box, is completely conserved, despite degeneracy in flanking sequences. Alignment was carried out manually. (B) Catalytic activity of UBE2W and UBE2WC91S in vitro. The GST fused proteins, UBE2W and FANCL, were purified from E. coli, and incubated with E1 activating enzyme, ubiquitin, and ATP. For negative controls, each component (except ATP) was omitted from the reaction, as indi-cated. The E2 (UbcH2), purchased from SIGMA, was used as positive control. The reactions were analyzed by Western blot with ubiquitin antibody (upper panel) or GST anti-body (lower panel). GST-UBE2W and ubiquitinated species are labeled with arrows.