Western Blot Analysis of Nav1.7 Protein

Cells or tumor specimens were lysed using radio-immunoprecipitation assay (RIPA) buffer with the addition of protease and phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations in each sample were quantified using a Pierce BCA Protein Assay Kit (Thermos Scientific, Waltham, MA, USA). Prior to performing gel electrophoresis, 1:1 of 2× Leammli Sample buffer (Bio-Rad, Hercules, CA, USA) was added to protein samples. The mixture was diluted with 5% 2-mercaptoethanol (ThermoFisher Scientific, Waltham, MA, USA). All protein samples were heated at 95 °C for 5 min and run on 4–15% Criterion TGX gradient gels (Bio-Rad). Gel transfer and immunoblotting detections were performed as previously described [57 (link)]. Primary antibodies for Na_V1.7 (EMD Millipore Corp., Darmstadt, Germany) were used at 1:1000, and the reference protein GAPDH (Cell Signaling Technology, Danvers, CA, USA) and β-actin (Cell Signaling Technology) were used at 1:2000. Horseradish peroxidase-conjugated anti-rabbit/mouse with a dilution of 1:1000 (Cell Signaling Technology) was used as secondary antibodies. The molecular weight-marker broad-range protein ladder (10–260 kDa) (Spectra Multicolor, ThermoFisher Scientific) was used to confirm the size of the protein of our interest.