2.5. Co-Immunoprecipitation Assay

Under basal condition, Raw 264.7 cells were plated in 6-well plates overnight. Under stress condition, Raw 264.7 cells treated with LPS (1 µg/mL) for 12 h. Then cells were washed with ice-cold PBS and lysed with IP lysis buffer (10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1% NP40, 150 mM NaCl, supplemented with protease and phosphates inhibitor cocktail). The lysates incubated with anti-iNOS antibody (1:100, Cell Signaling Technology) or p62 antibody (1:100, Sigma) overnight at 4 °C. 25 µL protein-G conjugated magnetic beads (Dynabeads Protein Immunoprecipitation kit, Thermo scientific) was added into each IP tube and incubate at 4 °C for 6 h. The supernatants were removed by aspiration and the beads were washed three times with the lysis buffer. Finally, the beads were denatured at 99 °C in 1×sample loading buffer and the IP products were detected by western blotting as mentioned above.