Ecl kit

Frozen tissues of mice were homogenized in RIPA buffer with phosphatase inhibitors. Western blot was performed as described previously (Kim et al., 2013 (link)). Homogenized proteins were quantified using Pierce™ BCA Protein Assay Kit (Thermo scientific, cat#23227). In brief, a total of 20 μg of proteins were separated by 10% SDS-PAGE, and transferred onto PVDF membrane (Bio-Rad). The membrane was incubated overnight with primary antibody, following the manufacturer’s instruction. All antibodies were purchased from Cell Signaling Technology (Danvers, MA); anti-β-Actin antibody (1:4,000, cat# 4,967), anti-STAT3 antibody (1:1,000, cat# 9,132), anti-phospho-STAT3 antibody (1:1,000, cat# 9,131), anti-AMPK antibody (1:2000, cat# 2,532), anti-phospho-AMPK antibody (1:1,000, cat# 2,531), anti-Akt antibody (1:1,000, cat# 9,272), anti-phospho-Akt antibody (1:1,000, cat# 9,275), anti-FoxO1 antibody (1:1,000, cat# 2,880), and anti-IκBα antibody (1:1,000, cat# 9,242) were used. After washing, the membrane was incubated in HRP-conjugated secondary antibody (1:1,000, Cell Signaling Technology) for 2 h at room temperature. Proteins were visualized using an ECL kit (Elpisbiotech, Daejeon, Korea). The intensity of the bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD), and normalized to that of an internal control.

The exosome pellets in ultracentrifugation tubes were lysed by the direct addition of RIPA buffer. After lysis, the protein concentration was quantified, and then we prepared western blotting samples with 20 μg of exosome protein. To detect proteins, the lysates were subjected to SDS-PAGE on 10 % acrylamide gels, with electrophoretic transfer to nitrocellulose membranes. The membranes were incubated with primary antibodies (1 : 1000) and secondary antibodies (1 : 5000), and proteins were visualized using an ECL kit (Elpis Biotech, South Korea). The following antibodies were used: mouse anti-TSG101 antibody (Abcam, UK), rabbit anti-CD81 (Abcam, UK), mouse anti-histone H2B (Abcam, UK), and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (heavy and light chain [H/L]; Invitrogen, USA) and goat anti-rabbit IgG (H/L; Abcam, UK).