Fig. 1. Simultaneous MCP-1 and RANTES co-activation of CCR2- and CCR5-expressing cells increases sensitivity of chemokine responses and promotes their heterodimerization. (A) CCR2b/CCR5 double-transfected HEK-293 cells were incubated with biotin-labeled mAbs against CCR2 and CCR5 or their respective isotype-matched control mAbs, followed by isothiocyanate-labeled streptavidin. (B) Ca²⁺ mobilization was induced by treatment with 10 nM MCP-1 or 10 nM RANTES in Fluo-3-loaded CCR2/CCR5-co-transfected HEK-293 cells. Results are expressed as a percentage of the chemokine-induced calcium response. Five experiments were performed; the figure depicts one representative experiment. Arrows indicate addition of stimulus. (C) Ca²⁺ mobilization was determined as in (B), following stimulation with different concentrations of MCP-1 or RANTES as indicated, added separately or simultaneously. Results are expressed as a percentage of the maximum chemokine-induced calcium response. The mean ± SD of four independent experiments is shown. (D) CCR2/CCR5-co-transfected HEK-293 cells were stimulated with chemokines (10 nM for 5 min at 37°C) and, where indicated, cross-linked with 1 mM DSS. Cell lysates were immunoprecipitated with anti-CCR2 antibody, electrophoresed and transferred to nitrocellulose membranes. The western blot was analyzed with anti-CCR5 antibody (left); as a positive control, unstimulated CCR2/CCR5-co-transfected HEK-293 cells were immunoprecipitated with anti-CCR5 antibody (lane 6). The membrane was stripped and reprobed with anti-CCR2 antibody as a control for protein loading (right). Arrows indicate the position to which monomers and dimers migrated.