Journal: PLoS Pathogens
Article Title: Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors
Figure Lengend Snippet: ( A ) The depicted constructs were used to generate transgenic animals that expressed human C4BP and/or FH in addition to their endogenous counterpart C4BP and FH molecules. ( B ) PCR analysis confirmed the presence of the either hu-C4BP α-chains (upper panel), human Factor H (FH) (lower panel) or both (both panels) in the tg animals, but not in BALB/c wt mice. (C) SDS-PAGE and western blot analysis confirmed that hu-C4BP ( C , upper panel non-reduced gel) and hu-FH ( C , lower panel, reduced gel) were detectable in the appropriately designated tg, but not in BALB/c mouse serum. ( D ) Serum levels of hu-C4BP and hu-FH were determined using a sandwich-ELISA. ( E) BALB/c (blue) and hu-C4BPxFH tg (red) serum (20%) deposit similar amounts of C3 on zymosan particles by flow cytometry. EDTA-treated BALB/c serum (negative control) did not deposit any C3 on zymosan (negative control; n = 3 sera from individual animals). Statistical analysis: Kruskal-Wallis analysis with Dunn’s post-test ( D ) and 1-way ANOVA with Bonferroni’s post-test ( E ).
Article Snippet: For flow cytometry analysis, the following antibodies were used: mouse anti human-C4BP MK104 either unconjugated or conjugated to biotin; mouse anti human-Factor H MRC OX24 unconjugated or conjugated to biotin; rabbit anti mouse-C4BP (homemade) conjugated to Dylight 647; mouse monoclonal anti mouse-Factor H (Hycult, HM1119) conjugated to biotin; goat anti mouse-C3c (Nordic Immunology, GAM/C3c/7S); anti mouse C3 FITC (MP Biomedicals #0855500) anti mouse Ly-6G brilliant violet 421 (BioLegend, #127627); anti mouse Ly-6C PerCP/Cy5.5 (BioLegend, #128011); anti mouse CD11c (BioLegend, #117317); anti mouse I-A/I-E brilliant violet 510 (BioLegend, #107635); anti mouse CD64 APC (BioLegend, #139305); anti mouse/human CD11b APC/Cy7 (BioLegend, #101225).
Techniques: Construct, Transgenic Assay, SDS Page, Western Blot, Sandwich ELISA, Flow Cytometry, Negative Control