Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis

Cells cultured in SDCA medium (2×10⁷ cells/ml) were subjected to alkaline trichloroacetic acid (TCA) lysis followed by SDS-PAGE and western blotting analysis [15]. For analysis of Ape1 maturation, SDS-PAGE was performed using 10% acrylamide gel. For analysis of Atg8-PE formation, a 13.5% acrylamide gel containing 6 M urea was used. For analysis of GFP-Atg8 cleavage, 12% acrylamide gel was used. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore) utilizing a semi-dry transfer apparatus (Bio-Rad) at 2 mA per 1 cm² for 45 min (10% or 12% gel) or 15 V constant voltage for 30 min (13.5% gel with 6 M urea). Following transfer, the membranes were blocked with 2% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 30 min at room temperature (RT). Membranes were then incubated with primary anti-Atg8 (1:5000) [6], anti-Ape1 (1:10000) [16] anti-GFP (1:10000; JL-8, Clontech) antibodies for 60 min at RT. Subsequently, membranes were washed three times with TBST and treated with horseradish peroxidase (HRP)-labeled anti-rabbit or -mouse secondary antibodies (Promega) at a dilution of 1:5000 for 30 min, followed by an additional wash cycle in TBST. Chemiluminescent signals generated with the enhanced chemiluminescence (ECL) reagent (GE Healthcare) were detected on an IR-LAS 1000 imaging system (FUJIFILM).