Anti phospho and total akt

Cells were lysed in NP-40 lysis buffer (20 mM Tris, HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 1% NP-40) containing protease inhibitors for 30 min at 4°C. Supernatant was collected following 10 min centrifugation at 16,000 g, 4°C, and protein content was quantified using the µBCA quantification kit (Thermo Fisher Scientific). Protein extracts (50 µg) were separated by SDS-PAGE on precast 4–12% acrylamide gel gradients and transferred to TransBlot Turbo Midi-size nitrocellulose membranes (Bio-Rad). Antibodies used were anti-LACC1 (E7 clone; Santa Cruz), anti-GAPDH, anti–IL-1β, anti-phospho and total Akt, AMPK, IKβ, IKKβ, and S6 from Cell Signaling Technology, anti-LC3B from Sigma-Aldrich, anti-p62 from Abcam, and anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:10,000; Jackson Immunoresearch), and revealed using the Chemiluminescence Western Lightening Plus Kit (Perkin-Elmer).