Pi32209

Proteins were separated using 4–12% gradient polyacrylamide SDS–PAGE gels (Life Technologies) and electrotransferred to 0.2 μM nitrocellulose membranes (GE Healthcare, Velizy-Villacoublay, France). After blocking in Tris-buffered saline with 0.1% Tween and 5% bovine serum albumin, membranes were blotted overnight at 4 °C with the appropriate primary antibodies. Primary antibodies were detected using the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (PI32209; Thermo Fisher Scientific, Bremen, Germany) with a Syngene camera. Densitometric analyses of immunoblots were performed using the GeneTools software.

Western blot analyses were performed as previously described (Lu et al., 2012 (link)). Mutant IDH1–R132H and total IDH1 were detected with rabbit monoclonal antibodies to IDH1–R132H (clone H09; 1:1,000; Dianova) and with rabbit monoclonal antibody to IDH1 (clone 2DH1; 1:1,000; Cell Signaling Technology), caspase 3 by rabbit polyclonal antibody (1:1,000; Cell Signaling Technology), Bcl-2 by rabbit polyclonal antibodies (1:1,000; Cell Signaling Technology), and actin by mouse monoclonal antibodies (1:5,000; Santa Cruz Biotechnology, Inc.). Standard secondary antibodies conjugated to HRP were used after incubation with primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (PI32209; Thermo Fisher Scientific) with a Syngene camera.