Anti nix

Western blotting was performed as previously described^{14 (link)}. Antibodies used in this study include: anti-PPTC7 (Novus Biologicals, catalog #NBP1-90654, 1:1000 dilution, 48 h incubation), anti-Citrate synthase (Cell Signaling Technologies, catalog #14309, 1:1000 dilution, overnight incubation), anti-beta actin (Abcam, catalog # ab170325, 1:1000 dilution, overnight incubation), anti-BNIP3 (rodent specific antibody, Cell Signaling Technologies, catalog #3769, 1:1000 dilution, 48 h incubation), anti-NIX (Cell Signaling Technologies, catalog #12396, 1:1000 dilution, overnight incubation), and anti-LC3B (Cell Signaling Technologies, catalog #3868, 1:1000 dilution, overnight incubation). For the immunoprecipitations, the following antibodies were used: anti-BNIP3 (Abcam, clone EPR4034, catalog #ab109362, WB 1:1000), anti-NIX (Cell Signaling Technology, clone D4R4B, catalog #12396, WB 1:1000); anti-FLAG (Sigma-Aldrich, catalog #SAB4301135, WB 1:1000), and anti-vinculin (SCBT, clone G-11; sc-55465, WB 1:1000).

Protein was extracted from the rat brain or the cultured cells with RIPA lysis buffer (Sigma-Aldrich) on ice. The supernatants were collected after centrifugation at 12000×g at 4°C for 20min. Protein concentration was determined using a BCA protein assay kit (Bio-rad, China), and the proteins were separated on SDS-polyacrylamide gels, and then transferred to a PVDF membrane. The membrane blots were first probed with a primary antibody. After incubation with horseradish peroxidase-conjugated second antibody, autoradiograms were prepared using the enhanced chemiluminescent system to visualize the protein antigen. The signals were recorded using X-ray film. Primary antibodies were rabbit anti-caspase 3, anti-Beclin-1, anti-LC3, anti-BNIP3, anti-NIX and anti-β-actin (Cell Signaling, San Jose, CA, USA). Secondary antibody is HRP-conjugated anti-rabbit (Jackson ImmunoResearch Labs, West Grove, PA, USA). β-actin was used as a protein loading control. The protein levels were first normalized to β-actin, and then normalized to the experimental controls. HIF-1a levels were determined by an ELISA kit (R&D System, Los Angeles, CA, USA).