Idh1 wild type

Approximately 1x10⁷ cells (cell number determined by visual counting in hemocytometer) were lysed using cell lysis buffer (Cell Signaling, MA, USA) containing protease inhibitors (Millipore, MA, USA). Protein concentration was estimated using the Bradford method in order to load equivalent amounts of protein for western blotting. Proteins were resolved using polyacrylamide gel electrophoresis (BioRad) under denaturing conditions, followed by transfer to PVDF membranes (Millipore, MA, USA). Membranes were blocked overnight in 5% milk (Santa Cruz Biotechnology, TX, USA) in TBST (Tris-buffered Saline with 0.1% Tween 20, pH 7.5) at 4°C. Membranes were then washed 3 times for 5 min each in TBST and incubated with primary antibodies diluted in TBST [IDH1 wild-type (Cell Signaling, MA, USA), IDH1 R132H mutant (Dianova, Hamburg, Germany), β- tubulin (Cell Signaling, MA, USA)] for 1 h at room temperature. Following 3 washes of 10 min each with TBST, HRP-conjugated secondary antibodies (IDH1 mutant (Abcam, MA, USA), IDH1 wild-type and β- tubulin (Cell Signaling, MA, USA) were added for 1 h in TBST at room temperature. Membranes were washed thrice in TBST as described above and developed onto autoradiographic film using an enhanced chemiluminescence substrate kit (Thermo Scientific, MA, USA).