Analysis of intracellular Nef protein expression in untreated or FK-treated Nef transfectants by (A) Western blot and (B) flow cytometric analyses. (A) JH6.2 (lane 1), J.Bru.2 cultured in the absence (lane 2) or presence (lane 3) of FK, and J.Bru.3 (lane 4) were 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 (logarithmic scale); y-axis, relative number of cells. A representative of four separate experiments is shown. The MFI (mean ± SD) of the four experiments expressed in AU was 3 ± 1 (JH6.2), 21 ± 3.5 (JBru.2), 254 ± 38 (JBru.2 + FK), and 322 ± 55 (JBru.3).