Quantitative Western Blotting Analysis

Cellular lysates were prepared and analyzed by Western Blotting as previously described [86 (link)]. Proteins were extracted by incubating the cells in lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 2 mM EGTA; 2 mM EDTA; 25 mM NaF; 25 mM β-glycerophosphate; 0.1 mM NaV; 0.2% Triton X-100; 0.3% Nonidet P40; proteinase inhibitor, Roche, Basel, Switzerland). Following centrifugation, protein concentrations of supernatants were determined by the BCA™ Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Next, 30–50 μg of protein per sample was separated electrophoretically using 10% SDS–PAGE gels and blotted onto Hybond™-C-Extra Nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). Proteins of interest were detected using the following antibodies: anti-phospho-IkBα (Ser32/36, mouse, monoclonal, 5A5, Cell Signalling, Danvers, MA, USA), anti-IkBα (rabbit, polyclonal, C-21, Santa Cruz, Singapore), anti-PCNA (mouse, monoclonal, PC10, Abcam), and anti-ubiquityl-PCNA (Lys164, rabbit, monoclonal, D5C7P, Cell Signaling). Chemiluminescence signals were detected on a ChemiDocMP System (BioRad, Hercules, CA, USA) using Clarity^TM Western ECL Substrate (BioRad) and Band intensities were quantified using ImageLab software 4.1. Intensity values of the protein of interest were normalized to the values of the corresponding loading control.