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. 2002 Nov 6;71(6):1285–1295. doi: 10.1086/344515

Figure 5.

Figure 5

Analysis of the cofactor activity of factor H in the proteolysis of fluid-phase C3b by factor I. a, Analysis by 8% SDS-PAGE under reducing conditions of the proteolysis of C3b by factor I in the presence of factor H. Factor H samples include purified factor H from two control individuals (N1 and N2), purified factor H from patients with aHUS (HUS2 [H2] and HUS3 [H3]), purified factor H corresponding to the wild-type (H29R) and mutated (H29C) alleles of patient HUS29, and purified recombinant wild-type factor H (r-factor H) and W1183L mutant factor H (r-W1183L) expressed in COS7 cells. Each panel includes three different concentrations of factor H. Identification of the C3b fragments is depicted in the panel corresponding to N2. The cofactor activity of factor H results in cleavage of the α′ chain of C3b in the fragments α65 and α45/α43. The top left panel corresponds to the control lanes of C3b alone and C3b plus factor I in the absence of factor H. b, Densitometric analysis of the proteolysis of C3b by factor I in the presence of factor H. Stained gels were scanned in a GS-800 Calibrated Densitometer (Bio-Rad), and the bands were quantified with ImageQuant, version 3.3 (Molecular Dynamics). The percentage of the C3b cleavage was determined as the ratio of the values corresponding to the α65 and the α′ bands. The code for the samples is indicated at bottom.