800cw goat anti rabbit igg

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.

Antibodies against human lactadherin (monoclonal sc-8029), CD63 (sc-5275 or ab68418), TSG101 (sc-7964), calnexin (sc-46669), GAPDH (sc-47724) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology, Inc. or Abcam. A policlonal antibody against lactadherin was also used (AF2767-SP) and purchased from RyD Systems. Antibody against human flotillin 1 (18634S) was purchased from Cell Signaling Technology (Danvers, MA). Alexa Fluor 488-conjugated anti-lactadherin antibody (sc-8029) was also purchased from Santa Cruz Biotechnology Inc. Anti-rabbit IgG-HRP (7074S) and anti-mouse IgG-HRP (7076S) secondary antibodies were purchased from Cell Signaling Technology. Goat anti-rabbit IgG-800CW (#92,532,211) and goat anti-mouse IgG-680LT (#925–68,020) secondary antibodies were obtained from Licor.

The procedure was followed, as previously described [37 (link)]. Briefly, after different stimulus cells were rinsed with cold PBS 1X, fixed with 4% (v/v) PFA, washed with 0.1 mM glycine, and blocked with 5% (v/v) horse serum for 30 min on ice. Cells were incubated with anti-GLUT4 (1/1000) and anti-ATP1A1 (1/2000) for 1 h on ice, followed by incubation with goat anti-rabbit IgG 800CW and goat anti-mouse IgG 680CW (LI-COR) secondary antibodies (1/10,000) for 1 h on ice. Fluorescence intensity was measured using the Odyssey CLx fluorescence imaging system (LI-COR) and quantified by densitometry using Image Studio Software (LI-COR). The content of GLUT4 in the PM was related to ATP1A1 protein. For other experiments, before stimulus, cells were pre-incubated for 30 min with 40 µM wortmannin or PD98059.

Cell protein extracts were prepared using RIPA lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mM sodium ortho-vanadate, and protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA)). Forty micrograms of protein extracts were separated by electrophoresis on 10% SDS-polyacrylamide gels [61 (link)] and transferred to a nitrocellulose membrane [62 (link)] (GE Healthcare Life Science, Amsterdam, The Netherlands). Nonspecific binding was blocked with 5% non-fat dry milk in a Tris-HCl-0.01% Tween 20 (TBS-T) buffer for 60 min at room temperature. The nitrocellulose membranes were incubated overnight at 4 °C with primary antibodies, and secondary antibodies raised in goat anti-mouse IgG 680CW and goat anti-rabbit IgG 800CW (LI-COR Biosciences, Lincoln, NE, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were developed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR) and were quantified by densitometric analysis using Image Studio Software (LI-COR).