Immunoblotting: A Comprehensive Protein Analysis

Immunoblotting was performed as previously described (13 (link)). Briefly, cell lysis buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 1 mM Na₃VO₄, 20 mM sodium pyrophosphate and Roche's complete protease inhibitor cocktail. The protein concentrations were measured by DC protein assay kit (Bio-Rad Laboratories, Inc.), and were separated by SDS-PAGE, then transferred onto ClearTrans Nitrocellulose Membrane (Wako). Membranes were blocked with 0.5 or 3% skim milk, and treated with primary antibodies in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). After treating with secondary antibodies in TBS-T, immunoreactive bands were detected using ECL Pro Western Blotting Detection Reagent (PerkinElmer.) and visualized using a LAS-3000 luminescent image analyzer (Fujifilm) or ImageQuant 800 (GE Healthcare). Valosin-containing protein (VCP) was used as a loading control.