Figure 2.

(A) Time course of serine/threonine phosphorylation of the CCR2B receptor in Mono Mac 1 cells. Mono Mac 1 cells were stimulated for the times indicated and lysed, and cell extracts were immunoprecipitated with an anti-CCR2 antibody. After SDS/PAGE and transfer, the same blot was first developed with a mixture of anti-phosphoserine/threonine antibodies (Upper) and then reprobed with the anti-CCR2 antibody MCP-1R05 (Lower). The molecular mass of the CCR2 receptor is 38 kDa. The figure is representative of three experiments with similar results. (B) Western blot analysis of the presence of GRK2 and GRK3 in Mono Mac 1 cell extracts (lane 1). For comparative purposes, the same analysis was performed in HEK293 cells transiently transfected with pcDNA3, GRK2, GRK3, GRK5, or GRK6 (lanes 2–6, respectively). Blots were tested with the anti-GRK2 AB9 (Left) and anti-GRK3 antibodies (Right). The molecular mass of GRK2 and GRK3 is ≈80 kDa and 78 kDa, respectively. (C) Western blot analysis of MCP-1-induced GRK2 translocation in Mono Mac 1 cells. Cells were stimulated with MCP-1 for the times indicated and particulate fractions were obtained as described in the text. Equal protein amounts were resolved in SDS/PAGE, blotted, and developed with anti-GRK2 AB9 antibody (Upper). Data were quantitated by laser densitometry, normalized by the signal of an unrelated band, and represented as the percentage of particulate GRK2 before stimulation (Lower). This experiment was repeated twice with similar results.