FIG. 4.

Subcellular localization of wild-type A3A and functionally defective mutants. APOBEC3 protein localization was determined by the indirect immunofluorescence of HA-tagged APOBEC3 proteins (HA) expressed in U2OS cells. Cells were costained with DAPI to visualize nuclei (Merge). U2OS cells were grown on glass coverslips and transfected with 0.8 μg APOBEC3 expression vector using Lipofectamine 2000 (Invitrogen). After 24 h, cells were fixed with 3% paraformaldehyde and extracted with 0.5% Triton X-100 in PBS. Cells were incubated with anti-HA MAb 16b12 (1:2,000) (Covance) and a 1:2,000 dilution of goat anti-mouse-conjugated Alexa Fluor 568 (Invitrogen) and DAPI (Sigma Aldrich). The coverslips were mounted in Fluoromount G (Southern Biotech), and images were acquired using a Leica TCS SP2 confocal microscope.