Transblotturbo midi size nitrocellulose membranes

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).

Protein was extracted from the cultured cells using RIPA Buffer (Fisher Scientific, Cat. no. PI89901) with protease/phosphatase inhibitors (Cell Signaling Technologies, Cat. no. 5872S) according to the manufacturer's instructions. Proteins were then resolved in 4%–20% or 4%–15% Mini‐PROTEAN TGX gels (BIORAD, Cat. no. 4561093 and Cat. no. 4561083, respectively) and transferred onto TransblotTurbo midi‐size nitrocellulose membranes (0.2 μm pore size, BIORAD, Cat. no. 1704271). The membranes were blocked for 30 min with LI‐COR Blocking Buffer‐PBS (LI‐COR, Cat. no. 4561083) and incubated overnight with primary antibodies and rocking at 4°C. The primary antibodies used were LAMP2A (Novus Biologicals, Cat. no. NBP267298, 1:1000 dilution) and β‐actin (Millipore Sigma, Cat. no. A5316, 1:4000 dilution). After overnight incubation, membranes were washed three times with PBS and incubated for 45 min with appropriate secondary antibodies conjugated with IRDye 680 or IRDye 800 dyes (LI‐COR, 1:2000 dilution). After washing with PBS, membranes were dried in the dark, scanned, and analyzed with an Odyssey IR imaging system (LI‐COR) and Image Studio Software.