Immunoblotting Protocol for Protein Detection

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).