20–40 µg of protein were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes (Invitrogen, CA, USA). The membranes were probed with antibodies against SIRT1, SIRT2 (Sigma-Aldrich), SIRT3, SIRT6 (Cell Signaling Technology), NRF2 (Abcam, cell signaling), HPRT, GAPDH (Proteintech), TBP, and β-actin (Abcam). HRP-conjugated donkey anti-rabbit and donkey anti-mouse were used as secondary antibodies (Jackson ImmunoResearch) and visualized by Pierce Super Signal Chemiluminescent Substrates.
For IP, cells or tissue were lysed using IP buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 5% glycerol), and cell extracts were incubated overnight with appropriate antibodies followed by incubation with protein A or G agarose beads for 4 hr at 4°C. After washing five times with IP buffer, immunocomplexes were resolved using SDS-PAGE and analyzed by western blot.
For IP, cells or tissue were lysed using IP buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 5% glycerol), and cell extracts were incubated overnight with appropriate antibodies followed by incubation with protein A or G agarose beads for 4 hr at 4°C. After washing five times with IP buffer, immunocomplexes were resolved using SDS-PAGE and analyzed by western blot.