Western blot analysis

Whole cell lysates were prepared as previously described [25]. Briefly, treated and untreated cells were extracted with lysis buffer (50 mmol/l Tris–HCl, pH 7.5, 5 mmol/l EDTA, 150 mmol/l NaCl, 0.5% Triton X-100, 10 mmol/l sodium fluoride, 20 mmol/l β-mercaptoethanol,womens silk pajamas, 250 μmol/l sodium orthovanadate, 1 mmol/l PMSF, and complete protease inhibitor cocktail; Sigma, St Louis, MO), and incubated at 4 °C for 30 min. The lysates were sonicated and centrifuged at 14,000x g for 15 min. The supernatants were collected and stored at −80 °C. Protein concentrations were determined by the BCA method. Protein (50 μg) was separated on 8-12% polyacrylamide-SDS gel and electroblotted onto nitrocellulose membranes (Bio-Rad laboratories, Hercules, CA). After blocking with TBS/5% skim milk, the membrane was incubated overnight at 4 °C with primary antibodies against HIF-1α (Cat: MA1-516; ABR, Rockford, IL) at concentration of 1:2,000 or polyclonal antibody against VEGF (Cat: sc-507; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) , at 1:200 followed by peroxidase conjugated anti-mouse IgG or anti-rabbit IgG for 1 hr at room temperature. Signals were detected with ECL. Data was analyzed using Un-scan-it gel analysis software (Silk Scientific, Orem, UT). Relative increase in protein expression compared to its own control is calculated.