Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV

Wilkins, Jordan; Zheng, Yi-Min; Yu, Jingyou; Liang, Chen; Liu, Shan-Lu

HEK293T cells were cotransfected with HIV-1-NL4.3 and increasing amounts of IFITM plasmids. pQCXIP-empty was used as a control and to ensure all transfections had an equivalent amount of total plasmid during transfection. At 48 hours post-transfection, HEK293T cells were harvested in RIPA buffer (containing 0.05% SDS). Cell debris was removed by centrifugation. A fraction of the lysate was used for total input analysis by Western blot while the rest was subjected to immunoprecipitation using anti-FLAG beads. Immunoprecipitations were incubated overnight at 4ËšC with rotation. Beads were centrifuged and washed three times with cold lysis buffer. All samples were boiled in sample buffer prior to resolving by SDS-PAGE. The gels were transferred to polyvinylidene difluoride (PVDF) membranes and blocked in blocking solution (5% non-fat milk in PBS 0.1% Tween-20) for 1 hour at room temperature. Membranes were washed (PBS with 0.1% Tween-20) three times followed by overnight incubation with the primary antibody in blocking solution overnight at 4ËšC. The membranes were washed three times and incubated with secondary antibodies conjugated to HRP for 1 hour at room temperature. The blots were incubated with HRP substrate (Millipore # WBLUR0100) and bands were visualized using a Fuji Imager.