Ligand binding and cofactor activity of the C3 proteins. Proteins were transiently expressed in 293T cells, concentrated and quantified before analysis (see Document S1). (A) Binding to MCP, factor H, factor B, and soluble CR1 (sCR1) in ELISA. For MCP and factor H, C3 protein was incubated at 15 ng/mL and for factor B, 200 ng/mL. Detection was made with chicken anti–human C3 and HRP-linked donkey anti–chicken IgY. * indicates a significant reduction in binding (P < .05). (B) Kinetic analysis of cofactor activity. C3 preparations were incubated for 0 to 30 minutes at 37°C with factor I and a cofactor protein (MCP, factor H, or sCR1). The zero control is before the addition of factor I. The last lane is an iC3b control. Samples were reduced and analyzed by Western blotting using either chicken anti–human C3 or goat anti–human C3 (insets) followed by an HRP-linked antiglobulin (see Document S1). Cofactor activity is assessed by the loss of the α chain and appearance of the α₁ and α₄₁ kDa major cleavage fragments. The minor α₄₃ kDa cleavage fragment is more variable but no pattern was observed that was specific for a mutant. Data are representative of 5 similar experiments.