Horseradish peroxidase conjugated goat anti rabbit or anti mouse secondary antibody

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.