Ecl reagent

The total protein of the cells was extracted using RIPA lysis buffer, and the protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher, Waltham, MA, USA). The proteins were separated using 10% SDS-PAGE gels and transferred to the PVDF membranes (Millipore, Chengdu, China). After blocking with 5% milk in Tris-buffer saline for 1 h, the membranes were incubated with primary and secondary antibodies and then visualized using enhanced chemiluminescence (ECL) reagents (VAZYME, Nanjing, China). The following primary antibodies were used: Akt (#9272, Cell Signaling Technology, Danvers, MA, USA), p-Akt (#9271, Cell Signaling Technology), and GAPDH (#8884, Cell Signaling Technology). ImageJ was used to calculate the level of target protein expression, with GAPDH as endogenous control.

Infected or transfected cells were washed three times with cold PBS and lysed with cold lysis buffer (1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF) in PBS) for 30 min. Lysates were clarified by centrifugation at 12,000× g for 10 min at 4 °C. Proteins in the lysates were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose membranes, and then probed with the antibodies indicated in the figure legends. Finally, the membranes were incubated with enhanced chemiluminescence (ECL) reagents (Vazyme, China).

The 10-week-old ovaries protein of wt and tg mice were lysed by cold RIPA buffer (Beyotime) for 30 min on ice, added to 5× SDS-PAGE loading buffer (Beyotime), and boiled at 100 °C for 10 min. Then, the lysates were separated by 8-12% SDS-PAGE and transferred electrophoretically onto PVDF membrane (Millipore, USA). After being blocked with 5% defatted milk powder in TBS-T buffer (20 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20), PVDF membranes were incubated with the primary antibodies (His-tag, Proteintech, Wuhan, China). The expression level of the Pai protein was marked by that of His-tag. After incubation with the HRP-conjugated secondary antibody (Santa Cruz, USA), the signals were measured using ECL reagents (Vazyme) using the Chemiluminescent Imaging System (Tanon, China). GAPDH (Proteintech) was used as an endogenous loading control.

Cells were washed three times with cold phosphate-buffered saline (PBS) and lysed in cold lysis buffer (1% Triton X-100, 1 mM phenylmethylsulfonyl difluoride [PMSF] in PBS) for 30 min. Lysates were clarified by centrifugation at 12,000 × g for 10 min. Proteins in the lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes (catalog number 10600001; GE Amersham), and then probed with the antibodies indicated in the figure legends. Finally, the membranes were incubated with enhanced chemiluminescence (ECL) reagents (Vazyme, China), and the signals were analyzed using an Amersham Imager 600 charge-coupled (device) CCD-based chemiluminescent analyzer (GE Healthcare). Proteins were detected with the following antibodies: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (10494-1-AP; Proteintech), anti-DDX5 antibody (10804-1-AP; Proteintech), anti-METTL3 antibody (15073-1-AP; Proteintech), anti-METTL14 antibody (26158-1-AP; Proteintech), anti-YTHDF2 antibody (24744-1-AP; Proteintech), anti-lamin B1 antibody (66095-1-Ig; Proteintech), anti-Flag monoclonal antibody (MAb) (F1804; Sigma-Aldrich), anti-MYC MAb (60003-2-Ig; Proteintech), and anti-HA MAb (H9658; Sigma-Aldrich). In addition, anti-NP antibodies were kindly provided by Chengjun Li (Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences).

The cultured podocytes were pretreated with lactacystin for 30 minutes, followed by high glucose treatment for 15 minutes. Cells were harvested after being washed 2 times with cold PBS and extracted in immunoprecipitation buffer (P0013, Beyotime) containing 0.5% PMSF, 1% protease inhibitor cocktail (HY-K0010, MedChemExpress), and 1% phosphatase I (HY-K0021, MedChemExpress), II (HY-K0022, MedChemExpress), and III (HY-K0023, MedChemExpress) inhibitor cocktails. Lysates (1 mg) were incubated with anti-PP2Acα (catalog ab106262, Abcam) at 4°C overnight. Protein A/G agarose beads were then incubated with the lysates for 1 hour at 4°C. Beads were washed with lysis buffer and PBS, resuspended in 35 μL of 2× SDS buffer, and boiled for 5 minutes. Samples were subjected to SDS-PAGE. Western blotting was performed by standard protocols using ECL reagents (Vazyme).

Cells were washed three times with cold phosphate-buffered saline (PBS) and lysed in cold lysis buffer (1% Triton X-100, 1 mM phenylmethylsulfonyl difluoride [PMSF] in PBS) for 30 min. Lysates were clarified by centrifugation at 12,000 × g for 10 min. Proteins in the lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes (catalog number 10600001; GE Amersham), and then probed with the antibodies indicated in the figure legends. Finally, the membranes were incubated with enhanced chemiluminescence (ECL) reagents (Vazyme, China), and the signals were analyzed using an Amersham Imager 600 charge-coupled (device) CCD-based chemiluminescent analyzer (GE Healthcare). Proteins were detected with the following antibodies: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (10494-1-AP; Proteintech), anti-DDX5 antibody (10804-1-AP; Proteintech), anti-METTL3 antibody (15073-1-AP; Proteintech), anti-METTL14 antibody (26158-1-AP; Proteintech), anti-YTHDF2 antibody (24744-1-AP; Proteintech), anti-lamin B1 antibody (66095-1-Ig; Proteintech), anti-Flag monoclonal antibody (MAb) (F1804; Sigma-Aldrich), anti-MYC MAb (60003-2-Ig; Proteintech), and anti-HA MAb (H9658; Sigma-Aldrich). In addition, anti-NP antibodies were kindly provided by Chengjun Li (Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences).

BCA assay kit (Beyotime, China) was used to measure the concentration of protein samples. The protein sample (20 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being electrically transferred onto PVDF membranes. After blocking with 30% bovine serum albumin (BSA) for 1 h at room temperature, the PVDF membranes were incubated with primary antibodies at 4°C overnight and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. ECL reagents (Vazyme, China) were used to stimulate chemiluminescent signals, which were measured and evaluated by the ChemiDoc XRS imaging system (Bio-Rad, United States).