Effect of eotaxin-3, MCP-1, and MIP-1β on ERK activation. Activated ERK was determined by Western blot analysis. Freshly isolated monocytes were stimulated with chemokines for the indicated times and phosphorylated ERK was detected with an antibody against diphospho-ERK. (A) Eotaxin-3 does not induce ERK phosphorylation. Cells were stimulated with MCP-1 (100 nM), MIP-1β (100 nM), or eotaxin-3 (300 nM) for the indicated times (upper blots). (B) Eotaxin-3 inhibits MCP-1–induced ERK phosphorylation. ERK phosphorylation in monocytes on MCP-1 (10 nM) stimulation for the indicated times is shown in the left panel. In the central and right panels the cells were treated with MCP-1 (10 nM) or eotaxin-3 (300 nM) for 3 minutes, and then stimulated with MCP-1 (10 nM) for 1 minute. To confirm equal loading, the blots were subsequently stripped and reprobed with an antibody directed against total ERK (lower blots).