FIG. 5.

Mapping of the lysine residues in IE2 that are conjugated by SUMO-1. (A) Total cell extracts were prepared from 293T cells transfected with 1 μg of vector alone (lane 1), or 1 μg of pJHA124 encoding wild-type IE2 (lane 2), pYX105 encoding IE2(K175R) (lane 4), pYX106 encoding IE2(K180R) (lane 6), or pYX104 encoding IE2(K175/180R) (lane 8) or the same four plasmids plus 2 μg of pJHA312 encoding Flag–SUMO-1 (lanes 3, 5, 7, and 9). Total cell extracts were prepared and subjected to electrophoresis on SDS–8% polyacrylamide gels, and immunoblot analysis was carried out with MAb CH810. (B) In vitro SUMO-1 conjugation assays. [³⁵S]methionine-labeled wild-type and mutant IE2 proteins were incubated at 37°C in the presence or absence of SUMO-1 modification reaction mixtures (Rxn Mix) as described in Materials and Methods. The reaction products were visualized by autoradiography. IE2-S, Flag–SUMO-1 conjugated IE2; ⧫, IE2 forms conjugated by an endogenous SUMO moiety.