Figure 1.
The cell surface exposure of eight different CKRs is reduced by HIV-1 Nef. (A) CHO cell lines, stably expressing the indicated human receptors, were transfected with bicistronic expression constructs encoding GFP alone or GFP together with HIV-1SF2 Nef. One day after transfection, cells were stained with either receptor-specific mAbs or the anti-HA-tag mAb, and steady-state surface levels of human receptors were quantified by flow cytometry. Dot plots of GFP expression (x-axis; FL-1 channel) relative to expression of the indicated receptors by using APC-conjugated mAbs (y-axis; FL-4 channel) are shown for viable cells, expressing GFP (left) or Nef/GFP (right). Fluorescence compensation adjustments were not required, because there is no cross-talk in the detection of GFP (FL-1) and APC (FL-4). (B) MFI for cell surface-exposed receptors was quantified on cells with medium GFP expression within the R3 gate (see A, top left for gating) relative to the MFI of GFP-negative cells in the R2 gate as described previously (Michel et al., 2005). Identical R2 and R3 gates were applied for the statistical analyses of all panels shown in Figures 1, 3, and 4. For each cell line, the ratio of the MFI of cells in R3/R2 takes into account variations in staining intensity that may exist between individual samples, and this ratio most accurately reflects the Nef-mediated down-regulation of the analyzed receptor. The arithmetic mean of R3/R2 ratios obtained from triplicates for CHO cell lines expressing GFP alone were arbitrarily set to 100%. Histogram bars represent the arithmetic means of triplicates + SD from one representative experiment of at least three independent experiments.