Analysis of intracellular Nef protein expression in untreated or FK-treated Nef transfectants by (A) Western blot and (B) flow cytometric analyses. (A) JBru.mut.8 cells were cultured in the absence (lane 1) or presence (lane 2) of FK and then subjected to Western blot analysis for Nef and tubulin. (B) Nef protein levels were determined by indirect immunofluorescence revealed by flow cytometry. Unshadowed histograms represent cells treated with anti-Nef MoAb + GAM-FITC, and shadowed histograms are negative controls (cells treated with anti-p66 of HCMV + GAM-FITC). X-axis, relative Nef expression detected by fluorescence intensity (log scale); y-axis, relative number of cells. A representative of four separate experiments is shown. The MFI of the four experiments (AU) was 4 ± 1.5 (JBru.mut.8) and 151 ± 29 (JBru.mut.8 + FK).