Immunoprecipitation. HeLa cells were cotransfected with pRSV/Rev and pOZ-Flag/NF90; 24 h post-transfection, the cells were lysed, α-Flag antibodies were added and the proteins precipitated by adding protein A agarose. The samples were analyzed by SDS-PAGE, and the proteins transferred to a PVDF membrane were immunodetected using α-Rev31–50 antibodies. Lane 1, extracts from HeLa cells cotransfected with both constructs. Lane 2, extracts from untransfected HeLa cells. Lane 3, same amount of HeLa cell extract prior to immunoprecipitation (input control). Arrow indicates the protein band (~28 kDa) corresponding to Rev.