Anti nbr1

Reagents and antibodies used were as follows: anti-Calnexin (#ab22595, Abcam, Cambridge, MA, USA), anti-LC3 (#L8918, Sigma-Aldrich, St. Louis, MO, USA), anti-NBR1 (#16004-1-AP, Proteintech, Rosemont, IL, USA), anti-PEX3 (#247042, Abcam), anti-PEX14 (#sc-23197, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PEX16 (#14816-1-AP, Proteintech, Rosemont, IL, USA), anti-PEX19 (#PA5-22129, Invitrogen, Carlsbad, CA, USA), anti-ABCD3 (#ab3421, Abcam, Cambridge, MA, USA and #SAB4200181, Sigma-Aldrich, St. Louis, MO, USA), anti-SQSTM1/p62 (#H00008878-M01, Abnova, Taipei, Taiwan), anti-VDAC (#55259-1-AP, Proteintech, Rosemont, IL, USA), anti-β-actin (#sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), and chloroquine (#C6628, Sigma-Aldrich, St. Louis, MO, USA).

The cell lysates were boiled at 95 °C for 3 min. 10–20 μg proteins were loaded onto 4–20% SDS-PAGE for separation and transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked by 5% skim milk and were probed by primary antibodies, anti-p62 (Cell Signaling Technology, Cat. 5114) (1: 500), anti-LC3 (Novus Biologicals, Cat. NB100-2331) (1: 1000), anti-decorin (Invitrogen, Cat. PA5-13538) (1: 100), anti-LAMP1 (Abcam, Cat. ab24170) (1:500), anti-p-TFEB S142 (Merck, Cat. ABE1971) (1:200), anti-TFEB (Bethyl Laboratories, Cat. A303-673A) (1:200), anti-NBR1 (Proteintech, Cat. 16004-1-AP) (1:200), anti-β-actin (Sigma-Aldrich, Cat. A2228) (1: 10,000) overnight at 4 °C. Next day, the nitrocellulose membranes were incubated with fluorescently labeled secondary antibodies (LI-COR Biosciences), Donkey anti-rabbit (Cat. 926-32213) (1: 10,000) or Goat anti-mouse (Cat. 926-68070) (1: 10,000) for one hour at room temperature. Images were acquired by a fluorescence imaging system (LI-COR Biosciences, Odyssey CLx) and were analyzed by Image Studio Lite (LI-COR Biosciences) software.

Following treatment with CP-31398, NBR1, or p62 immunoprecipitation was performed overnight at 4 °C using a classic IP kit (Pierce) and anti-p62 (cat #P0067; Sigma) or anti-NBR1 (cat #16004-1-AP; ProteinTech) rabbit pAbs, according to the manufacturer’s instructions. Immune complexes were eluted and washed before boiling in Laemmli buffer. Immunoprecipitates and cell lysates (input) were run in SDS-PAGE gel (4–20% precast gel, Biorad) and immunoblotted with HRP-conjugated anti-Bcl-2, anti-BclXL, anti-XIAP, anti-LC3, anti-NBR1, or anti-p62 Abs.