Elc western blotting detection reagents

Protein was extracted using RIPA Lysis buffer (Millipore, Temecula, CA) with protease inhibitor cocktail tables (Roche Diagnostics, Indianapolis, IN) or NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in 3% BSA/TBS-T, and the following antibodies were used: PhosphoPlus YAP (Ser127) Antibody Duet #46538 and Anti-rabbit IgG, HRP-linked Antibody #7074 (Cell Signaling Technology, Danvers, MA). Then, protein bands were examined using ELC Western Blotting Detection Reagents and the ImageQuant LAS 4000 mini (GE Healthcare, Backinghamshire, UK), and band intensities were quantified using ImageJ [43 ].

Equal amounts of protein extracts of the cells were separated by electrophoresed in 12% SDS-PAGE gels and transferred onto the polyvinylidene difluoride membranes (Thermo Fisher Scientific). The membranes were blocked with 5% BSA and 0.1% Tween-20 in Tris-buffered saline for 1 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: goat anti-galectin-1 (R&D), mouse anti-galectin-3 (Abcam), and rabbit anti-β-actin (Cell Signaling Technology, MA, USA). Membranes were washed; incubated with horseradish peroxidase-conjugated anti-goat IgG (R&D), anti-mouse IgG (Cell Signaling Technology), and anti-rabbit IgG (Cell Signaling Technology) secondary antibodies for 1 h at room temperature; and developed by ELC Western Blotting Detection Reagents (Amersham, GE Healthcare, UK). The blotted membranes were visualized using an ImageQuant LAS 4000 mini (GE Healthcare).