Anti beclin

EDHB was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in ethanol for biochemical assays. Anti-AKR1C antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-3 and anti-caspase-9 antibodies were procured from Enzo Life Sciences (NY). Anti-LC3A/B and anti-Beclin antibodies were purchased from Cell Signaling Technology (Boston, MA). Anti-β-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Anti-BNIP3 and anti-NDRG1 antibodies were purchased from Abcam (Abcam, MA).

Protein was extracted from the cultured cells with RIPA lysis buffer (1% NP40, 0.1% Sodium dodecyl sulfate (SDS), 100 μg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, in PBS) on ice. The supernatants were collected after centrifugation at 12000 × g at 4 °C for 20 min. Protein concentration was determined using a BCA protein assay kit (Bio-rad, China), and whole lysates were mixed with 4 × SDS loading buffer (125 mmol/l Tris-HCl, 4% SDS, 20% glycerol, 100 mmol/l Dithiothreitol (DTT), and 0.2% bromophenol blue) at a ratio of 1:3. Samples were heated at 100 °C for 5 min and were separated on SDS-polyacrylamide gels. The separated proteins were 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-β-catenin, anti-Beclin, anti-Atg7, anti-LC3 and anti-α-tubulin (Cell Signaling, San Jose, CA, USA). Secondary antibody is HRP-conjugated anti-rabbit (Jackson ImmunoResearch Labs, West Grove, PA, USA). α-tubulin was used as protein loading controls. The protein levels were first normalized to α-tubulin, and then normalized to control.

Cells were lysed in hypotonic medium (about 23 mM compared with 137 mM in isotonic medium) using a Dounce homogenizer, and nuclear fraction was obtained by centrifugation at 1,000g at 4°C. Proteins (20–30 μg) from lysates were subjected to electrophoresis (SDS-PAGE) and Western blotting analysis was performed as previously described [17 (link), 30 (link), 32 (link)]. To immunodetect endogenous HIF, rabbit polyclonal antibody against HIF-1α (1 : 1000; 132 KDa; Santa Cruz Biotechnology) was utilized. Rabbit polyclonal antibody anti-Bax (1 : 500; 21 KDa; Santa Cruz Biotechnology) and rabbit polyclonal antibody anti-Beclin (1 : 500; 52 KDa; Cell Signalling Technology, Danvers, MA, USA) were used to detect the proapoptotic and the autophagy markers, respectively. Bound antibodies were revealed by anti-rabbit immunoglobulin G1 conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and ECL reagents (Amersham Pharmacia Biotech, Italy). Anti β-actin (1 : 10,000; 42 KDa; Santa Cruz Biotechnology) was used to demonstrate the quality and equivalent loading of protein.

The EDHB chemical compound was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in ethanol for biochemical assays. Anti-NDRG1 and anti-BINP3 antibodies were purchased from Abcam (Abcam, MA). Anti-caspase-3 and anti-caspase-9 antibodies were purchased from Enzo Life Sciences (Life Sciences, NY). Anti-HIFα, anti-Beclin, anti-ATG12 and anti-LC3 antibodies were purchased from Cell Signaling Technologies (Danver, MA). Anti-AKR1C1, anti-Bcl-2, anti-p53, anti-TOM20 and anti-p21 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).