Figure 2

nIgG:ficolin immune complexes increase post-infection.

(a) Immunoblot analysis of total IgG (50 kDa heavy chain) and ficolin levels in the pooled sera of mice (n = 8) (top panel) and detection of nIgG (50 kDa heavy chain of mouse IgG3) and ficolin in nIgG:ficolin complexes (pulled down by Protein G beads) in serum (boxed in red), post-infection with 10⁶ or 10⁷ cfu P. aeruginosa over time course. The samples were derived from the same experiment, resolved under 12% reducing SDS-PAGE and the gels and blots were processed in parallel. Representative immunoblots (cropped for improving clarity and conciseness of presentation) from three independent experiments is shown. Full-length blots are presented in Supplementary Fig. 4a. (b) Immunofluorescence staining to detect co-localization of nIgG:ficolin complexes (using anti-mouse IgG3 and anti-ficolin) in spleen sections of WT mice (n = 3), infected with 10⁶ or 10⁷ cfu P. aeruginosa over time course. 100× objective. Scale bars, 10 μm. (c) Yeast two-hybrid screening to detect single chain interaction between nIgG and L-, H- and M-ficolin isoforms. nIgG interacts strongest with H-ficolin (red box).