Ecl buffer

Liver samples were homogenized in lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.9, 1 mM EDTA, 0.6% NP-40, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotonin, and 10 µg/ml trypsin inhibitor). Samples were then sonicated and incubated on ice for 30 minutes. Debris was removed by centrifugation at 10,000 rpm. Cellular total protein was extracted using a M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) Protein concentrations of each sample were determined in order to calculate the loading amount. Samples were separated in a denaturing 10% polyacrylimide gel and transferred to a 0.1 µm pore nitrocellulose membrane. Non-specific binding sites were blocked with Tris-buffered saline (TBS; 40 mM Tris, pH 7.6, 300 mM NaCl) containing 5% nonfat dry milk (Cell Signaling) for 1 hour at room temperature. Membranes were then incubated with appropriate primary antibodies in TBS with 0.1% Tween 20 (TBST). Membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase to show the bands with ECL buffer (GE Healthcare, UK). Parallel blotting of β-actin (Cell Signaling) was used as internal control.

To validate proteomics results, western blot analysis was performed. After the transfer of target proteins from SDS-PAGE, blocked membranes were incubated with Millipore anti-ROCK2 (Rho-associated protein kinase 2) (#07-1458), anti- IQGAP1 (Ras GTPase-activating-like protein 1) (#abt186), anti-PHB1 (#abn293), anti-PHB2 (#mabc953); Cell Signaling anti-GAPDH (#2118), anti-ILK1 (Integrin-linked protein kinase 1) (#3862), and Anti-Histone H3 (#4499) overnight at 4 °C. The membrane was rinsed with 1x TBS buffer prior to incubation with secondary antibody for 1 h. Blots were developed using ECL buffer (GE Healthcare) and the Geliance 600 imaging system (PerkinElmer).