FIG. 2.
Scatchard and competitive binding analysis of [125I]MIP1β onto HeLa-CD4/CCR5 clone JC.53. At the time of assay, the cells had grown to approximately 7 × 104/well. Specific [125I]MIP1β binding in counts/minute was calculated by subtracting the nonspecific binding (approximately 300 cpm) of [125I]MIP1β measured in the presence of 1 μM unlabeled MIP1β. Background binding of [125I]MIP1β, measured on HI-J cells lacking CCR5, gave binding values of approximately 300 cpm at all concentrations of unlabeled MIP1β. A Scatchard analysis of the binding data (inset) was performed to determine the number of CCR5 molecules/cell. The x intercept yields the number of accessible MIP1β binding sites/cell and equals approximately 1.45 × 105 CCR5 molecules/cell. The competition curve yields an 50% inhibitory concentration value of 15 nM, in close agreement with a KD of 17 nM which was obtained when the same binding data were used to generate a Scatchard plot with the x axis presented in molar concentration. Data points represent the means of triplicate assays.