Western Blotting for Protein Detection

Western blotting was conducted basically as reported¹⁵. Cells cultured in 6-well plates were washed twice with 2 mL iced-cold PBS, lysed by adding 100–200 μL of ice-cold lysis buffer [50 mM Tris-HCl (pH7.5) 150 mM NaCl, 1% Triton X-100] containing complete protease inhibitor cocktail (11873580001, Roche) and placed on ice for 20 min. Cell extracts were sedimented by centrifugation at 20,400 g for 10 min at 4°C. Protein concentration of supernatants was measured using the Protein Assay Bicinchoninate kit (06385–00, Sigma-Aldrich). Supernatants were mixed with 100–200 μL of sample buffer (2% SDS, 100 mM DTT, 50 mM Tris-HCl [pH 6.8], 5% glycerol, 0.001% bromophenol blue), incubated at 98°C for 5 min, and separated by SDS-PAGE. LC3 protein was detected using 15% polyacrylamide SDS-PAGE gels, which were made using the following regents: H₂O 1.4 mL, 1.5 M Tris-HCl (pH 8.8) 1.5 mL, 30% acrylamide/bis-acrylamide 3 mL, 10% SDS 60 μL, 10% ammonium peroxydisulfate: (APS) 50 μL, tetramethyl ethylenediamine: (TEMED) 5 μL. Stacking gel was prepared using the following reagents: H₂O 1.8 mL, 0.5 M Tris-HCl (pH 6.8) 750 μL, 30% acrylamide/bis-acrylamide 375 μL, 10% SDS 30 μL, 10% APS 30 μL, TEMED 5 μL). TFEB, S6K and 4EBP1 proteins detected using 10% polyacrylamide SDS-PAGE gels, which were made using the following reagents: H₂O 2.4 mL, 1.5 M Tris-HCl (pH 8.8), 1.5 mL, 30% acrylamide/bis-acrylamide 2 mL, 10% SDS 60 μL, 10% APS 50 μL, TEMED 5 μL. ULK1 protein detected using 7.5% polyacrylamide SDS-PAGE gels, which were made using the following regents: H₂O 2.9 mL, 1.5 M Tris-HCl (pH 8.8) 1.5 mL, 30% acrylamide/bis-acrylamide 1.5 mL, 10% SDS 60 μL, 10% APS 50 μL, TEMED 5 μL. The separated proteins were transferred to PVDF membranes (LC3: Immobilon-P, Merck Millipore; ULK1, TFEB, S6K, and 4EBP1: Hybond-P, Amersham) using transfer buffer (24 mM Tris base, 190 mM glycine, 20% methanol) by the wet transfer system (NA-1510B S/N 15J01, EIDO) at 150 mA for 1 hour. The membranes were blocked for 1 hour at room temperature in 1.0% skim milk in TBS-T (25 mM Tris base, 137 mM NaCl, 2.7 mM KCl, 0.16% HCl, 0.08% Tween 20, pH adjusted to 7.4). For phosphorylated proteins, 2.5% bovine serum albumin (A7906-50G, Sigma-Aldrich) in TBS-T was used as the blocking buffer. After blocking, the membrane was incubated for 1 hour with each primary antibody in blocking buffer at room temperature. The membrane was washed three times with TBS-T for 10 min and incubated at room temperature for 40 min with HRP-conjugated secondary antibody in blocking buffer, and then washed three times with TBS-T for 10 min. The membrane was incubated in the ECL Select western blotting detection reagent (GE Healthcare) for 5 min at room temperature. Signals were detected on a Gene Gnome-5 chemiluminescence detector (Syngene).