Anti vegf and anti β actin antibodies

After treatment, U87 MG cells were washed with PBS and lysed in ice-cold lysis buffer (25 mM HEPES, 1.5% Triton X-100, 0.1% sodium dodecylsulfate (SDS), 0.5 M NaCl, 5 mM EDTA, and 0.1 mM sodium deoxycholate) containing a protease inhibitor cocktail. Protein concentrations were quantified using a bicinchonic acid protein assay kit (Thermo, San Jose, CA). An equal amount of proteins from each group was separated using SDS-polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membranes. Membranes were incubated with a 5% skim milk solution (blocking solution) for 1 hour, and then incubated with anti-VEGF and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 16 hours. Membranes were probed with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, and then imaged using a Syngene G:BOX iChemi camera (Syngene, Cambridge, UK) and GeneSnap software (vers. 7.09, Syngene). β-actin was used as an internal control. The density of bands was determined with Gel-Pro Analyzer densitometry software.

Cells were homogenized with Mammalian Protein Extraction Buffer (GE Healthcare, Milwaukee, WI) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The total proteins (30 μg) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto membranes, and blocked with tris buffered saline containing Tween 20 in 5% non-fat dry milk. The membranes were incubated with anti-VEGF and anti- β-actin antibodies (Santa Cruz, CA, USA) and visualized by incubating with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) followed by Immobilon Western Chemiluminescent HRP Substrate (Millipore, St. Charles, MO). Chemiluminescence was detected by LAS-4000 (Fuji Film, Tokyo, Japan). The images derived from western blotting were analyzed through ImageJ (National Institutes of Health, Bethesda, MD) software for Windows.