Imagequant las 4000 mini image analyzer

Cells were lysed in SDS-PAGE protein loading buffer 1× (60 mM TrisHCl (pH 6.8), 10% glycerol, 2% SDS and 0.1% bromophenol blue) or assay buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and protease inhibitors). Proteins were analyzed by SDS-PAGE and transferred onto PVDF membranes, which were blocked with 3% BSA in PBS-buffered saline (PBS) containing 0.1% Tween 20 and incubated overnight at 4 °C with primary antibodies. After incubation with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature, membranes were revealed with the ECL system (Lumi-Light Western Blotting Substrate, Roche). Images were acquired by ImageQuant™ LAS 4000 mini Image Analyzer (Fujifilm, Tokyo, Japan) and quantified using ImageJ software. TUBULIN loading control was used when required. The primary antibodies used were the following: USP48 (Abcam, Cambridge, UK; ab72226, 1:1000), p65 (Cell Signaling Technology, Danvers, MA, USA; 8242, 1:1000), IκBα (Cell Signaling Technology, Danvers, MA, USA; 4814, 1:1000), P-IκBα (Cell Signaling Technology, Danvers, MA, USA; 2859, 1:1000), Cyclin E (Abcam, Cambridge, UK; ab2094; 1:1000), PCNA (Abcam, Cambridge, UK; ab29; 1:1000), NUCLEOLIN (Abcam, Cambridge, UK; ab70493; 1:1000); TUBULIN (Sigma-Aldrich, St. Louis, MO, USA; T5168, 1:1000).