Polyvinylidene difluoride nitrocellulose membranes

Following treatment of A549 and H1299 cells with B. nigra extract and/or CPT at the desired concentrations for 24 h, total proteins were extracted after washing with phosphate-buffered saline (PBS). The cells were lysed in ice-cold cytoskeletal lysis buffer supplemented with freshly prepared phosphatase and protease inhibitors (Roche, Mannheim, Germany). After total protein normalization, samples (50 μg protein) were loaded onto 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and separated electrophoretically, and the denatured proteins were transferred to polyvinylidene difluoride nitrocellulose membranes (Millipore, Darmstadt, Germany). At room temperature, the membranes were blocked in 5% dried non-fat milk for 2 h. After blocking, the membranes were incubated with primary antibodies overnight at 4 °C and followed by incubation with HRP-conjugated secondary antibodies [goat anti-rabbit IgG, 1:1000 or anti-mouse IgG, 1:1000] for 2 h at room temperature. The membrane blots were developed using chemiluminescence detection solution. GAPDH and β-actin were used as housekeeping controls.

Cells and tumor tissue were lysed with radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris–HCl (pH 7.4), 150 mmol/l NaCl, 5 mmol/l EDTA, 1% Triton X-100, 1% sodium deoxycholic acid and 0.1% SDS) plus with 2 mmol/l phenylmethylsulfonyl fluoride (PMSF), 50 mmol/l NaF, 1 mmol/l Na₃VO₄, and protease or phosphatase inhibitor cocktail (Sigma-Aldrich, Inc.). Soluble protein lysate concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of total or nuclear protein (20–120 μg) were resolved on 8–12% SDS-PAGE and transferred onto polyvinylidene difluoride nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were incubated in 5 % (w/v) bovine serum albumin (BSA/TBST) and incubated overnight at 4 °C on a shaker with specific primary antibodies. Membranes were washed with TBST and then incubated with secondary antibody (Sigma-Aldrich, Inc.) for 1 h at room temperature. After washing three times, the signal bands were visualized via chemiluminescence western blot detection reagent (ECL kit) and auto-radiographic film.

Cells were lysed with radioimmuno-precipitation (RIPA) buffer [(50 mmol/L Tris–HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 30 mmol/L Na2HPO4, 50 mmol/L NaF, 1 mmol/L Na3VO4, and protease or phosphatase inhibitor cocktail (Sigma-Aldrich, Inc. Shanghai, China)]. For xenografts, tumor issues well minced, and lysed with RIPA buffer. Samples were resolved on 8–12% SDS-PAGE and transferred onto polyvinylidene difluoride nitrocellulose membranes (Millipore, Billerica, MA). Membranes were incubated in 5% (w/v) bovine serum albumin (TBST/BSA) and stored overnight at 4°C on a shaker with specific primary antibodies (1/1,000 in TBST/BSA). Membranes were washed with TBST and incubated for 1 h with secondary antibody (1/10,000 in TBST/BSA) at room temperature. After several washes, the signals were visualized via the Odyssey western blot detection system.