Binding assay of radiolabeled human MIP-1α (A), RANTES (B), and MIP-1β (C) to CHEMR1. HEK293 cells were transfected with vector only, the CHEMR1 expression construct, or CCR-1 expression construct. After 24 hours, cells were harvested (about 2 × 106 cells) and incubated with 2 nmol/L 125I–MIP-1α, RANTES, and MIP-1α. The binding temperature for the MIP-1α was 37°C, whereas those for RANTES and MIP-1β were room temperature. The relative binding was obtained by dividing specific binding (bound cpm) of the radiolabeled chemokines to transfectants with the expression constructs by the specific-binding (bound cpm) to the mock-transfected cells. The average specific binding of 125I–MIP-1α to CCR-1 at 37°C was 14,000 cpm. The average bound cpm of 125I-RANTES to CCR-1 was 1,500 cpm. The average nonspecific binding of 125I–MIP-1α, RANTES, and MIP-1β to the mock-transfected cells was less than 800 cpm. Standard deviations were calculated by using three independent transfections and binding assays.
