Chemokine binding and functional assays.

(A) Competition binding curves were performed on CHO-K1 cell lines expressing wtCCR5 or 12L, L55Q, S215L, and ΔK228 mutants using 0.08 nmol/L [¹²⁵I]-MIP-1β as tracer. Results were analyzed by the Graphpad Prism software, using a single-site model, and the data were normalized for nonspecific (0%) and specific binding in the absence of competitor (100%). All points were run in triplicate (error bars: SEM). Data are representative of two independent experiments. (B) The functional response of the cell lines coexpressing apoaequorin and CCR5 natural mutants was tested following addition of MIP-1β. The luminescent signal resulting from the activation of the apoaequorin-coelenterazine complex was recorded for 30 seconds in a luminometer. Results were analyzed by nonlinear regression by using the Graphpad Prism software. The data were normalized for basal (0%) and maximal luminescence (100%). All points were run in triplicate (error bars: SEM). The displayed curves represent a typical experiment out of three performed independently. (C) The functional response to MIP-1β, MIP-1α, RANTES, and MCP-2 was tested on CHO-K1 cells expressing wtCCR5 or the A29S mutant using the aequorin assay. All points were run in triplicate (error bars: SEM). The displayed curves represent a typical experiment out of three performed independently. (D) Competition binding assay, using [¹²⁵I]-MCP-2 as tracer, and MIP-1α, MIP-1β, and MCP-2 as competitors, was performed on CHO-K1 cell lines expressing A29S. The data were analyzed and normalized as in Figure 3A. All points were run in triplicate (error bars: SEM). The displayed curves represent a typical experiment out of two performed independently.