Quantitative Western Blot Protocol for Protein Analysis

Tissue (1 × 2 mm) or cell line lysates were manually homogenized (PYREX homogenizer; VWR, Radnor, PA, USA) and prepared as previously described [32 (link)]. Supernatant protein was quantified (Pierce BCA protein assay kit; Thermo Scientific, Rockford, IL, USA), total protein lysates (15 μg) were denaturated in SDS sample buffer, separated on an SDS-PAGE gel [10% or 4%–20% (mTOR and 4E-BP1)], and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA; pore size 0.45 mm). Primary antibodies included anti-NAP1L1 (Abcam, Cambridge, MA, USA), anti-p57^Kip2, anti-menin-1, anti-phospho-ERK, anti-ERK, anti-phospho-mTOR, anti-mTOR, anti-phospho-S6K1, anti-S6K1, anti-phospho-4E-BP1, and anti-4E-BP1 (all from Cell Signaling Technology, Beverly, MA, USA). Immunodetection was performed using Western Lightning™ Plus-ECL (PerkinElmer, Waltham, MA, USA). Protein levels were confirmed with β-actin (Sigma-Aldrich, St. Louis, MO, USA). The optical density of the appropriately sized bands was measured using ImageJ software (NIH, Bethesda, MD, USA). The ratio between target protein expression and control protein was calculated.