Anti lamp2

Extracts for immunoblotting were acquired from N2a cells homogenized in RIPA buffer (radioimmunoprecipitation assay buffer, Thermo Fisher Scientific, Catalog Numbers 89900). Briefly, proteins were separated by electrophoresis and subsequently transferred to the PVDF membrane by electroblotting using standard protocols. The primary antibodies used were anti-LC3II (Cell Signaling Technology, catalog #2775s), anti-COX IV (Proteintech, catalog no. 11242–1-AP), anti-TOM40 (Proteintech, catalog no. 18409–1-AP), anti-MnSOD (Proteintech, catalog no. 24127–1-AP), anti-CLS (Proteintech, catalog no.14845–1-AP), anti-PLS3 (Proteintech, catalog no.28028–1-AP), anti-NDPK-D (Bioss, bs-11902R), anti-PINK1 (Novus Biologicals, catalog no. BC100–494), anti-Parkin (Abcam, catalog no. ab77924), anti-Drp1 (Cell Signaling Technology, catalog #8570), anti-Lamp2 (Cell Signaling Technology, catalog #49067), PGC1a monoclonal antibody (Proteintech, catalog no. 66369–1-Ig), recombinant anti-mtTFA antibody (Abcam, ab252432), GAPDH monoclonal antibody (Proteintech, catalog no. 60004–1-Ig), and anti-Tubulin (Proteintech, catalog no. 11224–1-AP).

Protein lysates were extracted from mouse liver, Hepa-1c1c7 cells, and donor liver with lysis buffer containing protease and phosphatase inhibitors at recommended concentration (Sigma-Aldrich). Aliquots containing 30 μg of proteins were loaded to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After transfer, membranes were blocked with 4% milk and incubated at 4 °C overnight with the following antibodies: anti-S6, anti-p-S6 (Ser235/236), anti-LAMP1, anti-LAMP2, anti-ATF4, anti-CHOP (Cell Signaling Technology, Denver, MA, USA), anti-LC3II (Novus Biologicals, Littleton, CO, USA), anti-β-actin, and anti-GAPDH (Abcam, Cambridge, MA, USA), respectively. Membranes were subsequently washed and incubated with horseradish peroxidase-conjugated secondary goat anti-mouse IgG or goat anti-rabbit IgG (Thermo Scientific, Rockford, IL, USA). The bound protein complexes were detected with enhanced chemiluminescence (Thermo Fisher Scientific, USA) and quantified by Image J (NIH, Bethesda, MD, USA).

HK‐2 cells (Human, kidney proximal tubular 2) were obtained from ATCC (ATCC^® CRL‐2190™). The primary antibodies used in this study include anti‐PARKIN (Cat no: #2132, 1:500; Cell Signaling Technology), anti‐p‐PARKIN (Cat no: orb312554, 1:250, Biorbyt), anti‐PINK1 (Cat no: #6946, 1:500; Cell Signaling Technology), anti‐BNIP3L (Cat no: GTX111876, 1:1000, GeneTex), anti‐BNIP3 (Cat no: GTX10433, 1:1000, GeneTex), anti‐HuR (Cat no: #12582, 1:1000, Cell Signaling Technology), anti‐TOMM20 (Cat no: ab56783, 1:200, Abcam), anti‐LC3 (Cat no: NB100‐2220, 1:200, Novus Biologicals), anti‐COXIV (Cat no: 4850, 1:1000, Cell Signaling Technology), anti‐LAMP2 (Cat no: 49067, 1:200, Cell Signaling Technology), and anti‐β‐ACTIN (Cat no: A1978, 1:1000, Sigma‐Aldrich). Secondary antibodies‐rabbit/mouse IgG HRP, Alexa Fluor 546 anti‐mouse IgG (H + L), Alexa Fluor 488 anti‐goat IgG (H + L), Alexa Fluor 647 anti‐rabbit IgG (H + L) were purchased from Invitrogen™. 2′,7′‐dichlorofluorescin‐diacetate (DCFH2‐DA), JC‐1 and DAPI (4′,6‐diamidino‐2‐phenylindole) mount were purchased from Sigma Aldrich. Magna RIP™ RNA‐Binding Protein Immunoprecipitation Kit was procured from Sigma‐Aldrich. The SuperSignal West Femto Maximum Sensitivity Substrate detection reagents were purchased from Thermo Scientific Inc.