Adipor1

BV2 cells were grown on poly-D-lysine-coated glass coverslips. After treatment, cells were fixed with 3.2% paraformaldehyde, then permeabilized in PBS pH 7.4, containing 0.3% Tween and 3% BSA. Cells were then incubated overnight with the appropriate primary antibody directed against CD11b (Abcam), iNOS (Merck Millipore), AdipoR1 or AdipoR2 (Sigma Aldrich) in the same buffer. At the end of the incubation time, cells were washed three times with PBS and incubated with either Alexa488-conjugated or Alexa594-conjugated secondary antibodies (Invitrogen) and Hoechst fluorescent stain (Invitrogen) for 1 h at room temperature. For NF-κB p65 subunit nuclear translocation study, cells were pre-treated with 10 μg/ml gApN or saline for 1 h before addition of 0.5 μg/ml LPS for 3 additional hours. Cells were then fixed and permeabilized as previously. Primary antibody against p65 was from Cell Signaling. Images were captured with an FV10i scanning confocal microscope (Olympus, France) and analyzed using Image J software (“National Institutes of Health” Image J Software™).