FIGURE 7.
N-terminal seven amino acid-mediated cleavage and secretion is required for the function of MCP-3. A, amino acid sequence of the N-terminal region of mouse MCP-3 and schematic of FLAG-MCP-3WT and FLAG-MCP-3Δ7NT. B, MC3T3-E1 cells were transfected with the indicated expression plasmids. After 12 h, the medium was replaced with serum-free fresh medium and incubated for 12 h. Then, the conditioned medium and cell lysate were analyzed by immunoblotting (IB) using the indicated antibodies. C, RT-PCR analysis of CCR1, CCR2, MCP-3, and GAPDH in RAW264.7 preosteoclasts and MC3T3-E1 osteoblasts. D, MC3T3-E1 cells were transfected with the indicated expression plasmids. After 12 h, the medium was replaced with serum-free fresh medium and incubated for 12 h. A RAW264.7 chemotaxis assay was performed using a Corning transwell unit. See “Experimental Procedures” for details.