Bicinchonic acid protein assay kit

Pro- and cleaved caspase-3 were analyzed using an immunoblotting method as described previously [42 (link)]. After drug treatment, human U87-MG-R9 cells were washed with PBS and lysed with an 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, including leupeptin (10 mg/mL), aprotinin (0.27 U/mL), and phenylmethylsulfonyl fluoride (PMSF, 100 mm). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Thermo, San Jose, CA, USA). Cellular proteins were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Following blocking with a 5% skim milk, the membranes were incubated with a caspase-3 antibody (Cell Signaling Technology, Beverly, MA, USA). Membranes were probed with the appropriate horseradish peroxidase-conjugated secondary antibodies. Levels of β-actin protein was immunodetected using a mouse monoclonal antibody (Sigma) as the internal standard. Immunoreactive proteins were detected using an enhanced chemiluminescence reagent (PerkinElmer, Waltham MA, USA) and then imaged using a digital analyzer (Syngene, Cambridge, UK) and a densitometry software (Syngene).

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.

The pathway of enzalutamide-induced apoptotic insults to human TMZ-sensitive U87MG and -resistant U87 MG-R glioblastoma cells was determined by analyzing the activities of caspases-8 and -9 as described previously [51 (link)]. Briefly, human U87 MG and U87 MG-R glioblastoma cells were exposed to 50 μM enzalutamide for 24, 48, and 72 h. Following drug treatment, cell lysates were prepared. Protein concentrations of cell lysates were measured using a bicinchonic acid protein assay kit (Thermo Fisher Scientific, San Jose, CA, USA). Activities of caspases-8 and -9 were measured using the metabolites of their specific peptide substrates IETD and LEHD, respectively, that were conjugated with 7-amino-4-trifluoromethyl coumarin to detect the fluorescent intensities. Cell lysates at 25 mg were incubated with 50 mM of these two fluorogenic IETD and LEHD substrates in a cell-free system buffer (200 μL). Activities of caspases-8 and -9 were determined by measuring their fluorescent intensities with a spectrophotometer.