Figure 1

AP activation was reduced by addition of heat-inactivated FB. Levels of C3a and Ba, generated in AP reactions containing purified proteins with increasing amounts of HFB, were measured by quantitative Western blot analysis. Reactions, labeled with boxed numbers 1–8, consisted of: (1) 10.4 ng/µl FB + C3 + FD (without HFB); (2) 10.4 ng/µl HFB + C3 + FD (without FB); (3) 10.4 ng/µl HFB + 10.4 ng/µl FB + C3 + FD; (4) 15.6 ng/µl HFB + 10.4 ng/µl FB + C3 + FD; (5) 20.8 ng/µl HFB + 10.4 ng/µl FB + C3 + FD; (6) 26 ng/µl HFB + 10.4 ng/µl FB + C3 + FD; (7) 31.2 ng/µl HFB + 10.4 ng/µl FB + C3 + FD; and (8) 10.4 ng/µl HFB + 10.4 ng/µl FB + C3, without FD. C3 concentration is 60 ng/µl and FD is 0.07 ng/µl in each reaction, except for reaction 8. (a) Representative blot of merged detection of C3/C3a using rabbit anti-C3a + donkey anti-rabbit IRDye-800 (green); and FB/Bb/Ba using goat anti-FB + donkey anti-goat IRDye-680 (red). C3a and Ba standards are in lanes 2–6, and protein ladders are in lanes 1 and 7. Graphs of reaction (b) C3a levels; and (c) Ba levels are means plus SD from 3–5 experiments. Values were not statistically different by 1-way ANOVA with Tukey’s multiple comparisons test. (d,e) Correlation plots of HFB concentrations and generated C3a, r² = 0.9224 (d); and Ba, r² = 0.9509 (e). Pearson’s coefficients of determination (r²) were calculated for HFB concentrations from 0 to 26 ng/µl.