Mouse anti galectin 3

Cell lysates were resolved by 8%–12% SDS-PAGE and transferred onto the PVDF membrane. Immunoblotting was carried out using the following antibodies: rat anti-galectin-3 (1:5000, Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA), goat anti-galectin-1 (1:5000, R&D systems), rabbit anti-FAK (1:5000), anti-phospho-FAK (Y397; 1:3000), anti-TNFα (1:3000), anti-IL6 (1:3000, Abcam, Cambridge, MA, USA), anti-integrin α3 (1:5000, Millipore), anti-NR1 (1:5000, Cell Signaling), mouse anti-galectin-3 (1:2000, Abcam), anti-actin (1:10,000, Millipore) and anti-Flag M2 (1:10,000, Sigma-Aldrich) antibodies. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat-anti rabbit, goat-anti mouse, goat-anti rat and donkey-anti goat IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Membrane was developed by reacting with chemiluminescence HRP substrate (Millipore) and exposed to the LAS-3000 image system (Fujifilm, Tokyo, Japan) for visualization of protein bands. The protein bands were quantified using the NIH ImageJ Software.

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).