Anti lamp2

Stable overexpression cells were grown to 90% confluence in 6-well plates and lysed in RIPA buffer supplemented with 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, and 1× protease inhibitor cocktail. The cell suspensions were incubated for 15 min on ice, followed by short sonication. The supernatants of the lysates were collected after centrifugation at 10,000 rpm for 10 min at 4 °C. The samples were heated with 1× SDS loading buffer at 98 °C for 5 to 10 min. Following 10% or 15% SDS–PAGE and wet blot analysis, the blots were probed with one of the following primary antibodies in TBST: anti-p-mTOR (phospho-S2448) (Bioworld, Nanjing, China, Cat. No. BS4706), anti-mTOR (S2442) (Bioworld, Cat. No. BS3611), anti-LC3 (Abcam, Cambridge, UK, Cat. No. ab51520), anti-p62 (Abcam, Cat. No. ab56416), anti-LAMP2 (Proteintech, Wuhan, China, Cat. No. 66301-1-Ig), or anti-GAPDH (Proteintech, Cat. No. 60004-1-Ig). The blots were incubated with horseradish peroxidase-conjugated secondary antibody after three washes, and the signals were visualized using a chemiluminescence (ECL) system (Engreen, Beijing, China, Cat. No. 29100).

Total protein was extracted from rat embryonic spinal cords (E11 and E12) or C17.2 cells using radioimmunoprecipitation (RIPA) buffer (Solarbio Science & Technology, Beijing, China). Protein concentrations were determined by a bicinchoninic acid assay (Solarbio Science & Technology). Proteins were separated by 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk solution, the membranes were incubated with primary antibodies at 4°C overnight. Following primary antibodies were applied in the experiment: anti-LC3 (Abcam, Cambridge, MA, United States; 1:1000), anti-LAMP2 (ProteinTech, Chicago, IL, United States; 1:1000), anti-Sirt1 (Cell Signaling Technology, Boston, MA, United States; 1:1000), anti-Bhlhe40 (ProteinTech, 1:1000), anti-PINK1 (ProteinTech, 1:1000), anti-VDAC1 (Abcam, 1:2000) and anti-GAPDH (ProteinTech, 1:5000). On the following day, the membranes were washed, and incubated with corresponding secondary antibodies (ProteinTech; 1:5000) at room temperature for 1.5 h. Enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, United States) was applied to detect protein-antibody interactions, which were visualized using a chemiluminescence detection system (C300, Azure Biosystems, United States).