The Evolutionary Dynamics of the Lion Panthera leo Revealed by Host and Viral Population Genomics

Antunes, Agostinho; Troyer, Jennifer L.; Roelke, Melody E.; Pecon-Slattery, Jill; Packer, Craig; Winterbach, Christiaan; Winterbach, Hanlie; Hemson, Graham; Frank, Laurence; Stander, Philip; Siefert, Ludwig; Driciru, Margaret; Funston, Paul J.; Alexander, Kathy A.; Prager, Katherine C.; Mills, Gus; Wildt, David; Bush, Mitch; O'Brien, Stephen J.; Johnson, Warren E.

Western blots using domestic cat and lion FIV as antigen were performed as previously described [46, 47] . The supernatant from virus-infected cells was centrifuged at 200 g for 10 min at 5uC. The resultant supernatant was centrifuged at 150,000 g at 4uC for 2 hours. Pelleted viral proteins were resuspended in 1/20 th of the original volume and total protein content was assayed using the Biorad Protein Assay. Twenty mg of viral protein were run on 4-20% Tris-Glycine gels and transferred to PDVF membranes (BioRad). Membrane strips were exposed 2-12 h to a 1:25 or 1:200 dilution of serum or plasma. After washing, samples were labeled with goat anti-cat HRP or phosphate conjugated antibody (KPL laboratories) at a 1:2000 dilution, washed, and incubated in ECL Western Blotting detection reagents (Amersham Biosciences) for 2 min, then exposed to Lumi-Film Chemiluminescent Detection Film (Boehringer Mannheim) or incubated in BCIP/ NBP phosphatase substrate (KPL laboratories) for 15 min [46] [47] [48] . Results were visualized and scored manually based on the presence and intensity of antibody binding to the p24 gag capsid protein.