Dynabeads protein immunoprecipitation kit

Chloroquine (CQ), SAR405, Torin 1, Rapamycin, Lipopolysaccharides (LPS), 1400 w dihydrochloride and Cycloheximide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitric oxide Griess reagent and RIPA buffer were obtained from Beyotime Biotechnology (Shanghai, China). Dynabeads protein Immunoprecipitation kit, TNF-α and IL-6 ELISA kit were from Thermo Fisher Scientific (Dreieich, Germany). The mouse monoclonal antibody against iNOS (Cat. No: 610328) was purchased from Biosciences (Heidelberg, Germany). The rabbit monoclonal anti-p62 for Western blot and immunofluorescence (Cat. No: ab109012) was purchased from Abcam (Cambridge, UK) and rabbit polyclonal anti-p62 for co-immunoprecipitation (Cat. No: P0068) was obtained from sigma. The rabbit polyclonal anti-LC3 (Cat. No: NB100-2220) for western blot was from Novus Biologicals (Littleton, CO, USA) and rabbit polyclonal anti-LC3 (Cat. No: PM036) for immunofluorescence staining was obtained from MBL International Corporation (Woburn, MA, USA). Anti-GAPDH (Cat. No: 5174) and anti-Lamp1 (Cat. No: 9091) were obtained from Cell Signaling Technology (Boston, MA, USA).

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.