Fig 1: Variants in CFB can affect its affinity for C3b binding and rate of C3 convertase decay in presence/absence of FH. (A) Schematic of recombinant FB proteins, blue arrows denoting the positions of specific mutants. (B) Western blot analysis of C3 convertase formation. Recombinant WT FB (FB his) shows decreased ability to form C3 convertase (lanes 3) as compared to WT FB (CA, lanes 1). There is no formation in presence of FH (lanes 2 and 4). Both p.Arg138Trp (lanes 5-6) and p.Phe286Leu (lanes 9-10) fail to form C3bBb in absence/presence of FH. p.Asp279Gly increases C3 convertase formation ~1.5-fold (lane 7) as compared to CA (lane 1) and is able to form C3bBb even in presence of FH (lane 8). Results were normalized to CA and presented as relative protein expression. Box plots show median, 1st and 3rd quartile ranges, and the individual data points of three independent experiments. Significance calculated for WT FB. (C) Western blot analysis of CA formed using WT FB or FB his, then decayed in presence/absence of FH. Decrease in C3 convertase at 0 and 2 min is observed as compared to WT FB. (D) Western blot analysis of CA formed using WT or p.Arg138Trp, then decayed in absence/presence of FH. No mutant convertase activity is observed at 0 min as compared to WT FB. (E) Western blot analysis of CA formed using WT or p.Asp279Gly, then decayed in absence/presence of FH. Decrease in natural and FH-mediated decay of p.Asp279Gly C3bBb is observed as compared to WT FB at all timepoints. (F) Western blot analysis of CA formed using WT or p.Phe286Leu, then decayed in absence/presence of FH. No mutant convertase activity is observed at 0 min as compared to WT FB. For all decay experiments, results were normalized to CA at 0 min and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Statistical significance shown for comparisons between CA/recombinant protein and CA+FH/recombinant protein+FH respectively at the same timepoint. Mean ± SD of three independent experiments. * P = 0.05, ** P = 0.01, *** P = 0.001, **** P = 0.0001.