M20005

Brain tissues from juvenile and aged SD rats were lysed in RIPA buffer (Solarbio, Beijing, China), following this protease and phosphatase inhibitors were added and then the sample was denatured at 100°C for 15 min. The protein samples were then separated using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Next, PVDF membranes were blocked with 5% skim milk powder solution for 1 h, incubated with primary antibodies, including anti-ACE2 antibody (1:1000, Proteintech, 21115-1-AP) and anti-Tubulin antibody (1:10000, Abmart, M20005) overnight, followed by secondary antibodies (1:5000) for 2 h at room temperature. The bands were visualised using an ECL kit chemiluminescence reagent (Billerica Millipore, MA, United States). Protein band signals were detected using the Chemidoc detection system (Bio-Rad, Hercules, CA, United States) and quantified by the ImageJ software (National Institutes of Health, United States).

Total cellular proteins were extracted with RIPA lysis buffer (Servicebio) and measured using the BCA protein quantification kit (Beyotime). Equivalent amounts of protein samples were loaded in each lane and separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Subsequently, the samples were transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with a primary antibody overnight at 4°C. The primary antibodies were as follows: MTHFD2 (1:5000, PHF6156, Abmart); PD‐L1 (1:500, T55980, Abmart); p‐JAK1 (1:2000, TP56310, Abmart); JAK1 (1:2000, T57173, Abmart); p‐STAT3 (1:1000, T56566, Abmart); STAT3 (1:2000, T55292, Abmart); Bcl‐2 (1:500, WL01556, Wanleibio); Bax (1:1000, WL01637, Wanleibio); Cyclin D1 (1:5000, 60186‐1‐Ig, Proteintech) and β‐Tubulin (1:5000, M20005, Abmart). Endogenous β‐tubulin was performed as an internal control protein for WB analysis. After washing with tris‐buffered saline with 0.1% Tween‐20 (TBST), the membranes were incubated with secondary antibodies for 1 h at room temperature. All bands were measured using an enhanced chemiluminescence (ECL) system kit (Servicebio) and analysed using ImageJ software. The statistical graphs about WB was presented in Figure S1.

The primary antibodies used are: rabbit anti-SCAMP5 (ab3432, Abcam;1:500 for WB); rabbit anti-LC3B (L7543, Sigma-Aldrich; 1:3000 for WB); mouse anti-p62/SQSTM1 (ab56416, Abcam; 1:2000 for WB); rabbit anti-TFEB (13372-1-AP, Proteintech; 1:1000 for WB); mouse anti-α-synuclein (610787, BD Biosciences; 1:1000 for WB); rabbit anti-huntingtin (5656P, Cell signaling; 1:1000 for WB); mouse anti-GM130 (610822, BD Biosciences; 1:300 for IF); mouse anti-Giantin (ab37266, Abcam; 1:300 for IF); rabbit anti-ERGIC53 (sc-66880, Santa Cruz; 1:100 for IF); rabbit anti-Calnexin (ab22595, Abcam; 1:2000 for WB); mouse anti-CD63 (ab8219, Abcam; 1:1000 for WB); mouse anti-DYKDDDDK-Tag (FLAG) (M20008, Abmart; 1:3000 for WB; 1:300 for IF; 1:200 for IP); goat anti-DYKDDDDK-Tag (FLAG) (NB600-344, NOVUS; 1:100 for IF); mouse anti-HA-Tag (M20003, Abmart; 1:200 for IP); mouse anti-GFP-Tag (M20004, Abmart; 1:2000 for WB); mouse anti-β-tubulin (M20005, Abmart; 1:3000 for WB); mouse anti-β-actin (M30002, Abmart; 1:2000 for WB).
The drugs used are: Rapamycin (553210, Merck, 100nM); Bafilomycin A1 (196000, Merck, 10-50nM); MG132 (474790, Merck, 3-5μM); Cycloheximide (C7698, sigma, 100μg/ml); Tetanus toxic (T3194. Sigma-Aldrich, 2nM); BrefeldinA (S1536, Beyotime, 2μM).