Chemiluminescent detection kit

5-6 × 10⁷ cells were washed twice with Phosphate-Buffered Saline (PBS) before being collected and lysed in lysis buffer (50 mmol/L Tris-Cl (pH 7.5), 150 mmol/L NaCl, 0.2 mmol/L EDTA, 1 mmol/L PMSF, and 1% (v/v) Nonidet-P40) for 30 min. The crude lysate was obtained by centrifugation at 13,200 rpm for 10 min at 4°C. 25 μg protein samples were separated by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Bio-Rad, Richmond, CA). After being blocked in 10% (w/v) nonfat milk powder (Sigma, St. Louis, MO, USA) at room temperature for 1 h, the membranes were incubated with antibodies against Eag1, VEGF, STAT3 (Abcam, Cambridge, MA), and GAPDH (Santa Cruz Biotechnology, CA, USA) overnight. Secondary antibodies were chosen according to the species of origin of the primary antibodies (Santa Cruz). Then the membranes were developed with chemiluminescent detection kit (Zhongshan Biotechnology, Beijing, China) and exposed to X-ray films.

A number of 5 × 10⁷–6 × 10⁷ cells were collected and lysed in ice-cold lysis buffer containing 50 mmol/L Tris-Cl (pH 7.5), 150 mmol/L NaCl, 0.2 mmol/L EDTA, 1 mmol/L PMSF and 1% (v/v) Nonidet-P40 for 30 min. The lysates were centrifuged at 13,200 rpm for 10 min at 4 °C and the supernatants were collected. Twenty five μg protein was resolved by a 12% SDS-PAGE and blotted on nitrocellulose membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked with 10% (w/v) nonfat milk powder at room temperature for 1 h, and then incubated with antibodies against Eag1 (Abcam, Cambridge, MA, USA), cyclin D1 (Cell Signaling Technology^®, Danvers, MA, USA), cyclin E and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then the membranes were developed with chemiluminescent detection kit (Zhongshan Biotechnology, Beijing, China) and exposed to X-ray films. Experiments were performed at least three times with representative data presented.

5-6 × 10⁷ cells were collected and lysed in ice-cold lysis buffer containing 50 mmol/L Tris-Cl (pH 7.5), 150 mmol/L NaCl, 0.2 mmol/L EDTA, 1 mmol/L PMSF, and 1% (v/v) Nonidet-P40 for 30 min. The lysates were centrifuged at 13,200 rpm for 10 min at 4°C and the supernatants were collected. 25 μg proteins were resolved by a 12% SDS-PAGE and blotted on nitrocellulose membranes (Bio-Rad). Membranes were blocked with 10% (w/v) nonfat milk powder at room temperature for 1 h and then incubated with antibodies against Eag1 (Abcam), p38 MAPK, phospho-p38 MAPK, and GAPDH (Cell Signaling Technology, Danvers, MA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Finally, the membranes were developed with chemiluminescent detection kit (Zhongshan Biotechnology). Experiments were performed at least three times with representative data presented.