Biotin-labeling Assay for Cell Surface Proteins

To determine the level of proteins at the cell surface we used a biotin-labeling protein assay (EZ-Link™ Sulfo-NHS-SS-Biotin [cat: 21331], Thermo Scientific, Rockford, IL, USA) as was previously described [20 (link)]. After stimulus, cells were incubated with a 0.12 mg/mL of biotin solution for 2 h at 4 °C, and then with 0.1 mM glycine solution for 30 min. The biotinylated proteins were pulled down using streptavidin-conjugated agarose beads (Pierce™ Streptavidin Agarose [cat: 20353], Thermo Scientific) for 2 h at room temperature. The biotinylated-plasma membrane proteins were eluted, then treated for Western blot and the nitrocellulose membranes were incubated with primary antibodies overnight at 4 °C and secondary antibodies were raised in goat anti-mouse IgG 680CW and goat anti-rabbit IgG 800CW (LI-COR) diluted 1/10,000 for 1 h at room temperature. The specific bands were developed using Odyssey CLx fluorescence imaging system (LI-COR) and were quantified by densitometric analysis using Image Studio Software (LI-COR). As the loading control of PM protein, total protein biotinylated-ATP1A1 and β-actin were used, respectively. Each biotinylated protein in the PM was related to biotinylated-ATP1A1 protein.