Analysis of C3b and C4b cofactor activity. Lysates of CHO cells expressing R69W, A304V, or wild-type MCP were incubated at 50 mM NaCl for 90 minutes with C3b or C4b and factor I. Cleavage of the α′ chain and generation of the α1 and α2 by the mutants are comparable with that of wild type. For the Western blot C3b cofactor activity (A), α′ and β represent the 2 chains of C3b. α1 corresponds to the larger proteolytic product of the α′ chain of C3b. The bottom portion of panel A is a 3-fold longer exposure of the same gel to show the smaller α2. SDS-PAGE, 10% gel, reducing conditions. Representative experiment of 3. For the Western blot of C4b cofactor activity (B), cleavage fragments C4d and α4 are similar between the mutants and wild type. The α3 is poorly biotinylated and not seen. The light band at approximately 60 kDa is most likely C4d (45 kDa) + α4 (15 kDa) and again is similar for the mutants in comparison with wild type. SDS-PAGE, 4% to 10% gel, reducing conditions. Representative experiment of 3.