Ab50339

The podocytes were plated in a 24-well plate. When 40% confluent, the cells were fixed, infiltrated and blocked. Each slide was incubated with anti-nephrin (ab216341, 1:500, Abcam, USA) and anti-podocin (ab50339, 1:500, Abcam, USA) at 4 °C overnight. Then the slides were washed three times with PBS and incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG (A0423, 1:100, Beyotime, Shanghai, China) for 45 min at room temperature. Finally, cells were observed under a confocal laser scanning microscope (Olympus, FV 1000, Center Valley, PA, USA).

Cultured cells were lysed and protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor (CW2200S, CWBio, China) and phosphatase inhibitors (CW2383S, CWBio, China). Equal amounts of proteins were resolved by 10% or 6% SDS-PAGE and were subjected to immunoblot analysis using standard methods with the following antibodies: NHPS2 (ab50339, Abcam), Synaptopodin (21064-1-AP), METTL3 (96391 s, Cell Signaling), YTHDC1 (77422 s, Cell Signaling), ADAR1(bs-2168R, Bioss), DNMT1 (ab188453, Abcam), WTAP (60,188–1-Ig, Proteintech) (all used at a 1:1000) and β-actin(AP0060, Bioworld) (used at 1:5000). The Secondary antibodies were purchased from Dingguo Biotechnology (Beijing, China). Semiquantitative analysis of the protein density by western blotting was performed using ImageJ (Version 1.5.3).

Paraffin-embedded kidney sections were incubated at 60 °C for 60 min, deparaffined with xylene three times for 15 min each, hydrated with gradient ethanol solution for 5 min each, and finally immersed in deionized water. Following a 20 min incubation at 95 °C with an antigen retrieval solution for antigen retrieval, the sections were let to cool naturally to room temperature. The endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ at room temperature for 10 min. The sections were then blocked with goat serum (ZLI-9056, ZSBIO company, Beijing, China) at 37 °C for 30 min, followed by incubation with anti-nephrin (1:2000, ab216341, Abcam), anti-podocin (1:1000, ab50339, Abcam) and anti-cleaved caspase-3 (1:400, 19677-1-AP, Proteintech, Wuhan, China) antibodies overnight at 4 °C. The sections were then washed in phosphate-buffered saline (PBS), treated for 20 min at room temperature with horseradish peroxidase-conjugated anti-rabbit secondary antibody (PV-9001, ZSBIO business), and colour development was performed by incubating with diaminobenzidine (DAB, ZLI-9018, ZSBIO company). Finally, the nuclei were stained with haematoxylin. At least five randomly chosen fields for each sample were evaluated under the microscope and analysed with Image-Pro Plus 6.0 software.

Frozen tissues were embedded in OCT, cut into 5 μm sections, and then stored at – 20 ℃. Rabbit anti-human Mfn2 antibody (1:100; Cat No. M6319, Sigma-Aldrich), rabbit anti-human nephrin monoclonal antibody (1:100; ab50339, Abcam), rat anti-human collagen IV alpha 5 monoclonal antibody (1:100; C-452, Cosmo Bio), rabbit anti-human Parkin antibody(1:100; 14060-1-AP, Proteintech) were reacted with renal tissue at 4 °C overnight. AF488-conjugated donkey anti-rabbit IgG antibody (1:500; A-21206, Invitrogen), and FITC conjugated donkey anti-rat IgG antibody (1:500, A-18740, Invitrogen) were incubated for half an hour at 37 ℃. Sections were observed using a fluorescence microscope (Nikon 80i; Nikon, Tokyo, Japan).