Chemiluminescence instrument

The sieving method was used to isolate glomeruli, as previously described [34 (link)]. Total protein was extracted from both cultured podocytes and isolated glomeruli using RIPA buffer containing a protease inhibitor cocktail (Sigma‒Aldrich, USA), PMSF (Beyotime, China), and a phosphatase inhibitor (Beyotime, China). After electrophoresis and electroblotting, the samples were incubated with primary and secondary antibodies and visualized on a chemiluminescence instrument (Bio-Rad, USA). The resulting bands were analysed using ImageJ. Western blotting was performed using the following antibodies: anti-ALCAT1 (1:1000, Invitrogen, USA), anti-β-actin (1:2000, Proteintech, China), anti-fission 1 (1:1000, FIS1; GeneTex, USA), anti-dynamin-related protein-1 (1:1000, DRP1; ImmunoWay, USA), anti-optic atrophy-1 (1:1000, OPA1; ImmunoWay, USA), anti-mitofusin2 (1:1000, MFN2; GeneTex, USA), anti-B-cell lymphoma-2 (1:1000, BCL2; Cell Signaling Technology, USA), anti-BCL2-Associated X (1:1000, BAX; Cell Signaling Technology, USA), anti-Caspase3 (1:1000, Cell Signaling Technology, USA), anti-PINK1 (1:1000, Novus, USA), anti-LC3B (1:1000, Abcam, USA), anti-P62 (1:1000, GeneTex, USA), anti-AMPK (1:1000, ImmunoWay, USA), anti-phospho-AMPK (1:1000, ImmunoWay, USA), and HRP-linked goat anti-rabbit/mouse IgG (1:10000, Antgene, China).

Total protein was extracted using the RIPA lysis buffer (Beyotime Biotechnology Inc., Shanghai, China) and quantified using the BCA reagent (Thermo Fisher Scientific). The denatured proteins were subjected to SDS/PAGE gel and transferred to PVDF membrane (Merck Millipore). The membrane was cut according to protein size and incubated with primary antibody: anti‐RAB1B (1 : 1000, ABclonal), anti‐PA2G4 (1 : 1000, ABclonal), anti‐SDF4 (1 : 1000, ABclonal), anti‐Flag (1 : 2000; Sigma‐Aldrich) or anti‐Tubulin (1 : 5000; Woburn, MA, USA). After washing with TBST solution, the bands were incubated with peroxidase‐conjugated goat anti‐mouse or anti‐rabbit IgG antibody (Proteintech, Wuhan, China) and the protein bands were developed using a chemiluminescence instrument (Bio‐Rad, Hercules, CA, USA).

Total protein of NPC cells was extracted using RIPA lysis buffer (Beyotime, China) and subjected to SDS-PAGE gel electrophoresis. The proteins were transferred into PVDF membrane (Merck Millipore, USA). The membrane was incubated with following primary antibody: anti-YBX1 (1:2000; Cell Signaling Technology, USA) or anti-GAPDH (1:5000; Cell Signaling Technology, USA). After incubated with secondary antibodies, the protein bands were defined by Chemiluminescence instrument (Bio-Rad, USA).

Total proteins from spinal cord tissue or bEnd.3 cells were extracted using RIPA lysis (Sigma, United States) and protease inhibitor (Sigma, United States). The tissue or cells were fully lysed after ultrasonic treatment for 120 s. Subsequently, the lysate was centrifuged at 4 °C and 12 000  r/min for 15 min, and the supernatant was collected. The protein concentration was determined using the BCA protein detection kit and a microplate reader (Thermo Fisher, United States). A 10% gel was utilized to separate proteins with different molecular weights. After adding samples and markers, the voltage was adjusted to 90 V for electrophoresis until the protein bands were completely separated. The proteins were then electro-transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 90 min. The PVDF membrane was blocked with 5% non-fat milk for 1.5 h and incubated with corresponding primary antibodies at 4 °C overnight. On the following day, the primary antibodies were removed, and the membrane was rinsed with TBST solution three times. Subsequently, secondary antibodies were added for incubation at room temperature for 1 h. Bands were covered with ECL chemiluminescence (Millipore, United States) and visualized using a chemiluminescence instrument (Bio-Rad, United States). Actin was used as an internal reference. Antibodies used are listed in Table S1.