Western blot analysis of CD14+ monocytes.

To define impairments in ERK signaling downstream of CX3CL1-CX3CR1 ligation in CD14⁺ monocytes from homozygous CX3CR1-M280/M280 donors relative to CX3CR1-WT/WT donors, 1.25 × 10⁶ cells were incubated in an ultra low attachment flat-bottomed 96-well plate (Costar) in RPMI 1640 containing 100 U/ml of penicillin and 100 μg/ml of streptomycin for 3 hours at 37°C. Recombinant human soluble CX3CL1 (R&D Systems, catalog 362-CX) was then added at a final concentration of 100 nM for 3, 10, or 30 minutes. Cells not exposed to soluble CX3CL1 were used as a control. After corresponding exposures to CX3CL1 or control buffer, the monocytes were washed with PBS, and the cells were resuspended in RIPA buffer (MilliporeSigma) containing Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). After 20 minutes of incubation at 4°C, cell lysates were clarified by centrifugation at 15,000 g for 10 minutes at 4°C. Supernatants were transferred to a new Eppendorf tube and stored at –20°C until further analysis. Western blot lysates were resolved on 10% SDS-PAGE gels, transferred onto PVDF membranes (MilliporeSigma, catalog IPVH304F0), probed with the indicated antibodies and developed with ECL (MilliporeSigma, catalog WBKLS0500) and film (Agfa X-Ray Film). Antibodies against p-ERK1/2 (Thr202/Tyr204; catalog 9101), p-AKT (Ser473; catalog 4060), and GAPDH (catalog 5174) were all obtained from Cell Signaling Technology. Goat anti-rabbit secondary Ab was from Southern Biotech (catalog 4010-05). Western blots were quantified by densitometry analysis using ImageJ analysis software (NIH) and Scion Image analysis software (Scion Corporation).