L7543

Western blot assays were performed according to previously described methods.¹¹ The membranes were probed with the following primary antibodies overnight at 4°C: anti‐cathepsin D (CTSD) (1:2000, Abcam, ab75852), anti‐lysosomal‐associated membrane protein 1 (LAMP1) (1:2000, Abcam, ab24170), anti‐phosphorylated protein kinase A (p‐PKA) (1:5000, Abcam, ab32390), anti‐PKA (1:2000, Abcam, ab75993), anti‐phosphorylated extracellular signal‐regulated kinase 2 (p‐ERK2) (1:1000, Abcam, ab201015), anti‐ERK2 (1:1000, Abcam, ab32081), anti‐TFEB (1:1000, Santa Cruz Biotechnology, sc‐48784), anti‐histone 2B (1:2000, Abcam, ab40886), anti‐LC3B (1:1000; Sigma, L7543), anti‐SQSTM1 (1:1000; Abcam, ab91526), anti‐Beclin1 (1:1000, Santa Cruz Biotechnology, sc‐11427), anti‐Atg5 (1:1000; Abcam, ab78073), anti‐phosphorylated TFEB (Ser142) (1:1000; Merck, ABE1971‐l), anti‐peroxisome proliferator‐activated receptor gamma co‐activator 1‐alpha (PGC‐1α) (1:1000; Abcam, ab54481) and anti‐β‐actin (1:5000, Abcam, ab8226). After incubation with horseradish peroxidase‐conjugated secondary antibodies, the membranes were visualized using SuperSignal Chemiluminescent Substrates (Thermo Fisher Scientific, 34080, Massachusetts, USA).

Cells were harvested and washed with cold phosphate-buffered saline (PBS). The proteins were extracted with RIPA Cell Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China) and maintained on ice for at least 30 min. The lysates were centrifuged at 12,000 g at 4 °C for 10 min, and the supernatant was transferred to a fresh tube. After the protein concentration was measured using the bicinchoninic acid (BCA) method, an equal quantity of total protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% non-fat dry milk powder in PBS for 1 h at room temperature and then incubated overnight at 4 °C with specific antibodies against LC3 (1:1000, Sigma, L7543), SQSTM1/p62 (1:1000, Abcam, ab56416), TFEB (1:2000, Bethyl Laboratories, A303–673A), LAMP-1(1:1000, Abcam, ab24170), and ACTB (1:5000, Sigma, A5441). After the membranes were incubated with the primary antibody, they were washed three times in TBST and then incubated with corresponding HRP-conjugated anti-mouse or anti-rabbit (Beyotime, A0208 or A0216) secondary antibody for 1 h at room temperature. Next, the membranes were washed and visualized using a Luminata Forte Western HRP Substrate (Merck Millipore, WBLUF0500). The bands were quantified with Image J software.

Neuro-2a cells were lysed in ice-cold RIPA Lysis Buffer. The protein concentration of each sample was determined by the bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). The samples were denatured by adding 5 × SDS-PAGE Sample Loading Buffer (Beyotime institute of biotechnology, China) and heating for 10 min at 100 °C. Equal amounts of protein were separated by10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred electrophoretically to polyvinylidene fluoride(PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline (50 mM TRIS-HCl, pH 7.5, 150 mM NaCl) containing 0.2% Tween 20, the membranes were probed with antibodies against the following proteins: LC3B (Sigma, L7543), SQSTM1/P62 (Abcam, ab56416), cleaved caspase-3 (CST, 9661), HIF-1α (Novus, NB100-105), PINK1 (Novus, BC100-494), BNIP3 (Abcam, ab109362), and β-actin (Proteintech, 60008-1-Ig). Following incubation with secondary anti-mouse (Proteintech, SA00001-1) or anti-rabbit (Proteintech, SA00001-2)antibodies, protein bands were visualized using enhanced chemiluminescence(ECL) blotting detection reagents (Millipore, WBKLS0500).