Western lighting plus

HCFs were made quiescent in serum-free DMEM/F-12 for 24 h, incubated without or with different concentrations of DDF at 37°C for the indicated time intervals. When inhibitors were applied, they were added 1 h except indicated before the exposure to DDF. After incubation, the cells were rapidly washed with ice-cold PBS and lysed with a sample buffer containing 125 mM Tris–HCl, 1.25% SDS, 6.25% glycerol, 3.2% β-mercaptoethanol, and 7.5 μM bromophenol blue with pH 6.8. Samples were denatured, subjected to SDS-PAGE using a 10% (w/v) running gel, and transferred to nitrocellulose membrane. The membranes were immunoblotted with one of the primary antibodies (1 : 1000 dilution) overnight at 4°C, followed by incubation with a peroxidase-conjugated secondary antibody at room temperature for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence reagent (Western Lighting Plus; Perkin Elmer, Waltham, MA, USA). The images of the immunoblots were acquired by using a UVP BioSpectrum 500 imaging system (Upland, CA, USA), and densitometry analysis was conducted using UN-SCAN-IT gel software (Orem, UT, USA).

Recombinant lectins (rHeltuba, rGal8N) were prepared using Escherichia coli as previously described (Tateno et al., 2011). Lectins were conjugated with HRP by using HRP labeling kit (Dojindo, Rockville, MD) at the concentration of 0.5 mg/ml and adjusted to the final concentration for incubation at 0.1 μg/ml.
One microgram of proteins from each cell population was separated by SDS‐PAGE on a 5‐20% gel (Perfect NT Gel system, NTH‐676HP, DRC, Tokyo, Japan) and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA). After blocking the membrane in Carbo‐Free blocking solution (Vector Laboratories, Burlingame, CA) for 1 hr at room temperature, it was incubated with HRP‐conjugated lectins overnight at 4°C. The signals were detected by using Western Lighting Plus (NEL104001EA, PerkinElmer). Lectin blot intensities were quantified using ImageJ software (National Institute of Health). The high‐intensity band was selected for quantification. Statistical significance was calculated by unpaired Student's t test (GraphPad Prism8 software).