FIGURE 3.

Competition binding assays in cells co-expressing CCR5 and CXCR4. A–D, competition binding assays were performed on cells expressing CXCR4 (A), CCR5 (C), or both receptors (B and D). Membranes were incubated with ¹²⁵I-SDF-1α or ¹²⁵I-MIP-1β as tracer and unlabeled MIP-1β (●), SDF-1α (○), TAK-779 (■), or AMD3100 (□) as competitors. The data were normalized for nonspecific binding (0%), in the presence of 300 nm SDF-1α (A and B) or MIP-1β (C and D), and specific binding in the absence of competitor (100%). All points were run in triplicates (error bars indicate S.E.). The displayed data are representative of two independent experiments. E–H, aequorin-based functional assay in cells co-expressing CCR5 and CXCR4. The functional responses of CHO-K1 cells expressing CXCR4 (E), CCR5 (G), or both receptors (F and H) were measured using the aequorin-based functional assay. E and F, cells were stimulated with SDF-1α (○), SDF-1α + AMD3100 (□), or SDF-1α + TAK-779 (■). G and H, cells were stimulated with MIP-1β (●), MIP-1β + AMD3100 (□), or MIP-1β + TAK-779 (■). Luminescence was recorded for 30 s. The results were normalized for base-line activity (0%) and the maximal response obtained with 25 μm ATP (100%). The displayed data are representative of three independent experiments. All points were run in triplicates (error bars indicate S.E.).