FIG. 3.

Time course analysis of factor I cofactor activity of SPICE, VCP, and the single-amino-acid substitution mutants of VCP for human complement protein C4b. Cofactor activity was determined by incubating human C4b with VCP, SPICE, or the mutants in the presence or absence of human factor I. The reactions were stopped after the indicated time periods by adding sample buffer containing dithiothreitol, and C4b cleavages were visualized by subjecting the samples to SDS-PAGE analysis on an 11% gel. During C4b cleavage, the α′-chain is cleaved into N-terminal 27-kDa, C-terminal 16-kDa, and central C4d fragments; generation of these fragments indicates the inactivation of C4b. The amount of α′-chain left was assessed by measuring its intensity by densitometric analysis and is represented graphically in the lower panel.