Western Blotting

Western blotting was undertaken according to the method described by Ogasawara et al. [23] with slight modifications. Powdered samples of 5 days OL were weighed and homogenized using shaking machine (Beads Crusher μT-12; Taitec, Koshigaya, Japan) with zirconia beads (3 mm × 3 pieces) in ice-cold homogenization buffer at 3,200 rpm for 1 min. The homogenization buffer contained 100 mM Tris-HCl (pH 7.8), 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, protease inhibitor cocktail (Complete Mini; Roche, Basel, Switzerland), and phosphatase inhibitor cocktail (PhosSTOP; Roche). Homogenates were centrifuged at 15,000 × g for 15 min at 4°C and supernatants collected. Protein concentrations of supernatants were determined using a protein quantification kit (Protein Assay Rapid Kit; Wako Pure Chemical Industries, Osaka, Japan). Samples were mixed with ×3 sample buffer (1.0% v/v 2-mercaptoethanol, 4.0% w/v SDS, 0.16 M Tris-HCl (pH 6.8), 43% v/v glycerol, and 0.2% bromophenol blue) and heated at 95°C for 5 min. Samples with 50 μg of total protein were electrophoresed using polyacrylamide gel (gradient, 4%–20%), and transferred to polyvinylidene difluoride (PVDF) membranes. After transfer, membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and blocked with 5% skimmed milk in TBST for 1 h at room temperature. Membranes were then washed and incubated overnight with primary antibodies at 4°C. Primary antibodies (Cell Signaling Technology, Danvers, MA, USA) used were: phospho-p70S6 kinase (Thr389), total p70S6 kinase, and total ribosomal protein S6 (rpS6). After reactions with primary antibodies, membranes were washed and incubated with horseradish peroxidase-conjugated anti-rabbit antibodies for 1 h at room temperature. After washing, chemiluminescent reagents (Luminata Forte; Millipore, Bedford, MA, USA) were used to facilitate detection of protein bands. Images were scanned using a chemiluminescence detector (GeneGnome; Syngene, Cambridge, UK). After measurements, membranes were stripped using stripping buffer (Restore™ Plus; Pierce, Rockford, IL, USA) and stained with Coomassie Brilliant Blue (CBB) to verify equal loading in all lanes [24]. Using captured images, band intensities were measured using ImageJ. For direct comparison, samples from the eight experimental conditions were run on the same gel.