BiFC assays with CD1 and CD2 mutants of A3G. (A) BiFC assays with CD2 mutants of A3G. All co-transfections included mRFP expressing plasmid, and RFP expression was used to identify transfected cells (panels labeled RFP). (B) BiFC and immunofluorescence assays with CD1 mutants of A3G. Expression of the CD1 mutants was verified by detection of the H65R-NY, H65R-CY, C97S-NY, and C97S-CY proteins in transfected cells by immunofluorescence. An anti-A3G polyclonal antibody produced in rabbit was used as a primary antibody and Alexa Fluor 568-conjugated goat antibody to rabbit IgG (H+L) (Molecular Probes) was used as secondary fluorescent antibody. (C) Comparison of BiFC and protein expression between WT A3G and H65R mutant A3G. Western blotting analysis of lysates of cells co-transfected with A3G-NY and -CY (I, 0.25 μg DNA each) or H65R-NY and -CY (II, 1 μg DNA each). The A3G proteins were identified by using a polyclonal anti-A3G antibody, and the same lysates were analyzed by using an anti-tubulin antibody to ensure that equivalent amounts were loaded onto gels. (D) Western blotting analysis of lysates of 293T cells and viral lysates produced from cells transfected with CD1 mutants F70A-NY, F70A-CY, Y91A-NY, and Y91A-CY. The cell lysates were also analyzed using anti-tubulin antibody to insure equivalent loading of cell lysate proteins (panel labeled α-tubulin). (E) BiFC assays with CD1 mutants F70A and Y91A.