Specific modulation of CCR5 cell-surface expression by CD4. (A) CHO-K1 cells were cotransfected with fixed concentrations of a plasmid encoding CCR5-Luc and increasing concentrations of the CD4-YFP plasmid or controls. CCR5 surface expression was determined by FACS analysis using a mouse anti–human CCR5 (2D7) as primary antibody followed by a Cy5-conjugated anti–mouse antibody. Representative experiments are shown. Above each panel, the YFP-associated signal (between parentheses) of the indicated YFP-fused protein is shown, using the same units as in panel B. (B) Results of 14 independent experiments, each representing 8 to 10 individual transfections. The luciferase signal was used to monitor the total amount of CCR5. Experiments in which the luciferase signal was different from the average by more than 10% were discarded. Surface CCR5 values, represented as the percentage (± SEM of 4-6 grouped values) of control cells expressing CCR5 alone, were plotted as a function of the YFP signal, reflecting the concentration of CD4-YFP, of control leptin receptor fused to YFP (OBR-YFP) or free YFP. *The experimental conditions used for quantification of CD4 and CCR5 (see Figure S4A). (C) Coimmunoprecipitation analysis of CD4-CXCR4 interaction. CHO-K1 cells were cotransfected with plasmids coding for CD4 and CXCR4-YFP and processed for immunoprecipitation as described in Figure 2D. (D) Absence of CXCR4 cell-surface modulation by increasing concentrations of CD4. The experiment was conducted as described in panel A, using a mouse anti-CXCR4 antibody (12G5) followed by a Cy5-conjugated anti–mouse antibody. Each point corresponds to an individual transfection.