PolyP enhances the capacity of C1-INH to dampen C1s-mediated cleavage of C4 and C2 in a concentration- and size-dependent manner. SDS-PAGE analysis under reducing conditions was used to evaluate the effect of polyP concentration and size, on C4 and C2 cleavage. Concentrations of polyP reflect the concentration of the monomer. (A) C4 alone is shown in lane 1. C1s alone is not detectable at the concentration used. C1-INH and C1s were reacted with C4 in the presence of varying concentrations of polyP130 or 500 μM of monoP (lane 5) as shown and described in supplemental Materials and methods. C4 cleavage products were detected by Coomassie staining after SDS-PAGE. (B) C1-INH and C1s were reacted with C2 in the presence of varying concentrations of polyP130 or 500 μM of monoP (lane 5). (C) C1-INH and C1s were reacted with C4 in the presence of 500 μM monoP (P1), P3, polyP14 (P14), polyP60 (P60), or polyP130 (P130) for 30 minutes, after which C4 cleavage products were visualized by SDS-PAGE. Gels are representative of experiments performed a minimum of 5 times.
