Figure 1. Mouse MCP1 C-terminus decreases the affinity of human MCP1 to CCR2.

A. h-MCP1 decreased membrane-bound CCR2 on microglia. Primary microglia activated overnight with 100ng/ml LPS were treated with saline (Ctr) or 10nM recombinant hMCP1 or hc-MCP1 for 1 h and then swelled in hypotonic buffer for 20 min to cause cell lysis. The plasma membrane sheets that adhered to the coverslips were stained for Mac-2 (Green) to visualize the membrane sheets and CCR2 (Red). Microglia immunostained with secondary antibodies only showed no fluorescence. Scale bar = 5 μm. The fluorescent intensity was quantified using ImageJ program and the fluorescent intensity was normalized to plasma membrane area. 20 cells per condition were used and 3 separate experiments were performed. The results were analyzed using student’s t-test. *p<0.05. B. h-MCP1 has higher affinity for CCR2. Microglia cells (CHME3) were incubated with 50nM biotinylated human MCP1 in the presence or absence of a gradient concentration of unlabeled h- or hc-MCP1 for 1 hour at 4°C. After further incubation with FITC-Avidin for 30 minutes at 4°C, the cells were washed and FITC signal was quantified using a plate reader. Background signal was measured using biotinylated soybean trypsin inhibitor (supplied by R&D) and subtracted from the experimental raw data. Then the FITC signal was normalized to total protein concentration. The final data were expressed as percentage of fluorescence in the absence of unlabeled MCP1. The data were expressed as Mean ± SD (n=4). *p<0.05, compared with Ctr; #p<0.05, comparison between h-MCP1 and hc-MCP1.