Immunoblotting was performed as previously described (19 (link)). Cells were lysed with RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) supplemented with a protease and phosphatase inhibitor cocktail (Nacalai Tesque). Cellular proteins were quantified using a DC protein assay kit of Bio-Rad Laboratories (Richmond, CA, USA). Equal amounts of proteins were loaded onto the gels, separated by SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). These membranes were probed with first antibodies (Abs) such as anti-LC3B antibody (Ab) (Novus Biologicals LLC, Littleton, CO, USA), anti-p62 (D-3) monoclonal (m) Ab (sequestsome-1), anti-β actin (C4) mAb, anti-GAPDH (6C5) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anticleaved-caspase-3 Ab, and anti-PARP Ab (Cell Signaling Technology, Danvers, MA, USA). Immunoreactive proteins were detected using horseradish peroxidase-conjugated second Abs and an enhanced chemiluminescence reagent (ECL) (Millipore). Densitometry was performed using a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories).