(A) Biphasic thymocyte binding of¹²⁵I-MIP-1β. Initial binding of ¹²⁵I-MIP-1β to thymocytes is competed by increasing concentrations of unlabelled MIP-1β. Scatchard analysis of all points indicate a total Kd value of 35 nmol/L with an estimated 4,500 sites per thymocyte over the entire population (not shown). If each phase of the reaction is analyzed separately, a Kd value of 0.1 nmol/L (first phase; dotted line) and 35 nmol/L (second phase) are determined. This reaction carried out at 4°C contains 70,000 cpm of input ¹²⁵I-MIP-1β per reaction, and is representative of n = 5 experiments each using thymocytes from different donors. (B and C) Heterologous competiton of¹²⁵I-MIP-1β bound to thymocytes by MIP-1α and RANTES, respectively. This result is taken from the same experiment as depicted in (A). (D) Ligand binding study using ¹²⁵I-MIP-1β and CCR-5/NIH 3T3 transfectants. ¹²⁵I-MIP-1β was added at 120,000 cpm per reaction. (E) and (F) Heterologous competition of¹²⁵I-MIP-1β bound to CCR-5/NIH 3T3 cells by MIP-1α and RANTES, respectively.