Anti bak antibody

The Bak oligomerization assay was performed as previously reported47 (link). Briefly, mitochondria were isolated and incubated with 4 mM disuccinimidyl suberate (Sigma, St. Louis, Missouri, USA) for 30 min at room temperature. Cross-linked samples were analyzed by western blotting using an anti-Bak antibody (Epitomics, Burlingame, California, USA). Rabbit anti-VDAC1 monoclonal antibodies (Abcam, Cambridge, Massachusetts, USA) were used as a mitochondrial loading control.

Anti-PDK1, anti-HIF-1α, anti-c-Myc and anti-Bax antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-Bak antibody from Epitomics (Burlingame, CA, USA) for western blot analysis. The cryopreserved tumor tissues were homogenized and lysed. Protein lysates were prepared in a buffer containing complete protease inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN, USA). After lysis, the protein content was quantified using Bradford's method. Equal amounts of protein were loaded onto each lane of a polyacrylamide gel for SDS-PAGE. The proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA) and incubated with 10% nonfat milk for 1 h. After three washes, the membranes were incubated with the indicated primary antibody overnight. After another three washes, the membranes were incubated with secondary horseradish peroxidase-conjugated goat anti-mouse antibodies (Millipore). The membranes were washed three times and visualized using an ECL system (GE Healthcare, Wauwatosa, WI, USA) with BioMax Light film (Kodak, Rochester, NY, USA). To quantify the signal intensity, Quantity One 1-D analysis software (Bio-Rad Laboratories, Hercules, CA, USA) was used.