Western Blot Protein Quantification

Cells (5×10⁵ or 1×10⁶) were lysed in protein lysis buffer (2% SDS, 50 mM Tris-HCl, pH 7.5) containing protease inhibitor and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) 26 (link). Protein concentration was determined by using a Bradford assay kit (BioRad, Hercules, CA, USA). An amount of 50-80 µg protein lysates were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes, which were blocked with 5% milk in Tris-buffered saline (TBS) for 1 h at room temperature, and then incubated with primary antibody overnight at 4 °C. After washing with TBST buffer (TBS with 0.05% Tween X100), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) for 2 h at room temperature and then revealed using enhanced chemiluminescent (ECL) reagent (Advansta, San Jose, CA, USA). Image and emission signal density measurements were quantified by using a BioSpectrum Image System (UVP, Upland, CA).