Anti idh2

Cells and tissues were washed three times with cold 1x PBS, harvested, and lysed for 30 min in 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% glycerol with protease inhibitors (BioTool) and TSA (Sigma), then quantified by Bradford assay and immunoblotted with: anti-MnSOD (1:1000 dilution, Millipore, #06–984), anti-MnSOD-K68-Ac (1:1000 dilution, Abcam,  #Ab137037), anti-MnSOD-K122-Ac (1:500, Abcam, #Ab214675), anti-SIRT3 (1:1000 dilution, Cell Signaling, #D22A3), anti-IDH₂ (1:1000 dilution, Cell Signaling, #56439), anti-IDH2-K413-Ac (1:1000 dilution, Epitomics, Inc, Burlingame, CA (this company has been bought by Abcam, Inc.)), anti-OSCP (1:1000 dilution, Santa Cruz Biotechnology, #sc-365162), anti-OSCP-K139-Ac (1:1000 dilution, Epitomics, Inc, Burlingame, CA), and anti-actin (1:10,000 dilution, Cell Signaling, #4970). Secondary antibody includes anti-rabbit and anti-mouse (1:10,000 dilution, Cell Signaling, #7074, #7076). For the MnSOD tetramerization assay, lysed cells were treated with 0.1% glutaraldehyde for 10 min at room temperature before samples were immunoblotted with anti-MnSOD antibody.

Whole-cell lysates were obtained using PRO-PREP protein extraction solution (Intron Biotechnology, Gyeonggi-do, Korea). Proteins were separated by SDS-PAGE, and bands were transferred to nitrocellulose membranes (Pall Corporation, Pensacola, FL, USA). The membranes were then incubated with the following primary antibodies at 4°C overnight: anti-p53, anti-Idh2, anti-B-actin, anti-Cdk1, anti-Cdk2, anti-Cdk4, anti-Cdk6, anti-iNOS, anti-Caspase3, and anti-PARP (Cell signaling); anti-p16 and anti-p21 (Abcam, Cambridge, UK); anti-COX-2 (SantaCruz); and anti-Prx-SO3 (Ab Frontier, Seoul, Korea). After washing with buffer, the membranes were incubated with the appropriate secondary antibody (Thermo Scientific) at room temperature for 6 hours.

The cell protein was extracted using RIPA buffer (Thermo). Protein concentration was determined by BCA Protein Assay (Beyotime). Total protein was loaded, fractionated by SDS-PAGE, transferred to PVDF membrane, and probed with anti-β actin (Abcam), anti-PKM (Abcam), anti-PFKM (Cell Signaling Technology), anti-PDHB (Abcam), and anti-IDH2 (Cell Signaling Technology). Signal was detected using Chemiluminescence imaging system (Tanon, Shanghai, China).