Anti vdac1

For immunoblotting, samples resolved by SDS-PAGE were transferred onto 0.2 μm nitrocellulose membranes (Bio-Rad, cat# 1620112). Membranes were blocked with 5% non-fat milk, and then incubated with the appropriate primary antibodies: anti-HA (Sigma-Aldrich, cat# H3663), 1:5000; anti-Flag (Sigma-Aldrich, cat# F1804), 1:5000; anti-ICDH (Xu et al., 2010 (link)), 1:10,000; anti-tubulin (DSHB, E7) 1:10,000; anti-PDHA1 (ProteinTech, cat# 18068-1-AP), 1:5000; anti-ATPB (ProteinTech, cat# 17247-1-AP), 1:2000; anti-TOM20 (ProteinTech, cat# 11802-1-AP), 1:5000; anti-VDAC1 (ProteinTech, cat# 55259-1-AP), 1:2000; anti-Cyto c (Santa Cruz Biotechnology, cat# sc-13560), 1:1000; anti-Calnexin (ProteinTech, cat# 10427-2-AP), 1:2000; anti-ANT1/2 (ProteinTech, cat# 17796-1-AP), 1:1000; anti-GM130 (BD Biosciences, cat# 610822), 1:2500; and anti-ADPR (Sigma-Aldrich, cat# MABE1016), 1:1000. Membranes were then incubated with an appropriate IRDye infrared secondary antibody and scanned by using an Odyssey infrared imaging system (Li-Cor’s Biosciences).

TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N‐Acetyl‐L‐cysteine (NAC) were purchased from Sigma‐Aldrich. MitoTracker™ Red FM (M22425), Hoechst 33342 (H1399) and anti‐Ubiquitin WB Antibody (13–1600) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) (1:1,000). Anti‐phospho‐Ubiquitin (Ser65) (ABS1513‐I) was purchased from Merck KGaA company (1:1000). Anti‐P53 (21891–1‐AP), anti‐PINK1 (23274–1‐AP), anti‐Parkin (14060–1‐AP), anti‐Drp1 (12957–1‐AP), anti‐Opa1 (27733–1‐AP), anti‐Mfn1 (13798–1‐AP), anti‐Mfn2 (12186–1‐AP), anti‐Caspase‐9 (10380–1‐AP), anti‐BAX (50599–2‐Ig), anti‐Caspase‐3 (19677–1‐AP), anti‐VDAC1 (10866–1‐AP), anti‐Lamin B (12987–1‐AP), anti‐Cytochrome c (10993–1‐AP), and anti‐β‐actin (60008–1‐Ig) were purchased from ProteinTech Group, Inc., (Chicago, IL, USA) (1:1,000). Anti‐γ‐H2A.X (ab81299) was purchased from Abcam (1:1000). Anti‐AMPKα (2532) and p‐AMPKα (50081) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA) (1:1000). Anti‐phospho‐DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti‐phospho‐DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. (1:1,000).

Cells were lysed in RIPA lysis buffer and the protein concentration of the cell lysates determined by BCA Protein Assay (Thermo Scientific, 23227). Equal amounts of protein were loaded onto SDS-PAGE gel and transferred to PVDF membranes. Western blotting was performed using primary antibodies and secondary antibodies conjugated with HRP. For immunoblotting, the following antibodies were used: anti-SENP2 (Abcam, ab58418, 1:1000), anti-HIF-1α (Cell Signaling Technology, 79233, 1:1000), anti-phospho-threonine (Cell Signaling Technology, 9386, 1:1000), anti-Ubc9 (Cell Signaling Technology, 4786, 1:1000), anti-BrdU (Cell Signaling Technology, 5292, 1:1000), anti-ubiquitin (Cell Signaling Technology, 3936, 1:1000), anti-cleaved-caspase 3 (Cell Signaling Technology, 9661, 1:1000), anti-SUMO1 (Cell Signaling Technology, 4930, 1:1000), anti-SUMO2/3 (Cell Signaling Technology, 4971, 1:1000), anti-SENP1 (Cell Signaling Technology, 11929, 1:1000), anti-SENP3 (Cell Signaling Technology, 5591, 1:1000), anti-HA-Tag (Cell Signaling Technology, 3724, 1:2000), anti-Flag-Tag (Cell Signaling Technology, 14793, 1:2000), anti-hexokinase 1 (Proteintech, 19662-1-AP, 1:1000), anti-hexokinase 2 (Proteintech, 22029-1-AP, 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:1000), anti-alpha tubulin (Proteintech, 66031-1-Ig, 1:5000), anti-GAPDH (Proteintech, 10494-1-AP, 1:5000).