Polyvinylidene fluoride membrane

MSFs seeded in two 12-well plates were collected after being treated with 10 ng/mL Na₂S₂O₄ for 6 h and 100 μM atropine for 24 h in sequence. Total proteins were extracted from MSFs using radioimmunoprecipitation assay (RIPA) lysis buffer (CWBio, Beijing, China) containing a 1% protease inhibitor cocktail (Sigma-Aldrich). After incubation on ice for 30 min, debris was removed by centrifugation (13,000 rpm) at 4°C and protein concentration was quantified by BCA assay. Proteins were diluted in 5× loading buffer and denatured at 98°C for 3 min 20 μg protein samples were fractionated by SDS-PAGE using the commercial polyacrylamide gel (Bio-Rad, Hercules, CA, United States) and transferred onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA, United States). Membranes were blocked in 5% nonfat dried milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T) at room temperature for 1 h. Subsequently, membranes were incubated with the indicated primary antibodies against phosphorylated endothelial NO synthase (p-eNOs, phospho T495; 9574, rabbit monoclonal antibody, 1:1000; Cell Signaling Technology, Danvers, MA, United States), β-catenin (ab32572, rabbit monoclonal antibody, 1:1000; Abcam, Cambridge, MA, United States), ZO1 (5406, rabbit monoclonal antibody, 1:1000; Cell Signaling Technology, Danvers, MA, United States), P53 (9282, rabbit monoclonal antibody, 1:1000; Cell Signaling Technology, Danvers, MA, United States), and GAPDH (AF5718, goat polyclonal antibody, 1 mg/mL; R&D Systems, Minneapolis, MN, United States) overnight at 4°C. After washing with 1 × TBS-T for 30 min, membranes were incubated with corresponding secondary antibodies (1:10000; ZSGB-Bio) at room temperature for 1 h. Bands were visualized with a Fluor ChemE (ProteinSimple) and ImageJ was used to analyze the gray value of each protein band.

Total RNA from cell samples was extracted using the RNAiso Plus reagent (#9108, TaKaRa), and cDNA was synthesized using the Prime Script RT Reagent Kit (#RR047A, TaKaRa). For RT‐qPCR analysis of selected gene expression, the TB Green Premix Ex Taq II (#RR820A, TaRaKa) was employed, with GAPDH serving as the endogenous control. The QuantStudio5 real‐time PCR system (Applied Biosystems) was used to measure relative mRNA expression levels, with data analysis performed using the 2^{(−ΔΔC(T))} method. Each sample was examined in triplicate, and the experiment was repeated independently three times. Primer sequences are provided in Table S3.
Cell proteins were extracted using RIPA lysis buffer (#P0013B, Beyotime), and the protein concentration was determined using a BCA protein assay kit (#WB6501, NCM Biotech). Equal amounts of proteins were isolated using SDS‐PAGE and blotted onto a polyvinylidene fluoride membrane (Millipore, USA), followed by blocking with 5% nonfat milk for 1.5 h at room temperature. Membranes were then incubated overnight with the designated primary antibodies at 4°C, followed by incubation with the secondary antibody for 1 h. Signal detection was performed using the enhanced chemiluminescence (ECL) detection kit (#P2200, NCM Biotech) with an automatic chemiluminescence/fluorescence image analysis system (Bio‐Rad, USA). The antibodies used are listed in Table S4.

Protein lysates (30 µg) were prepared using the radioimmunoprecipitation assay lysis buffer (Rockland, Philadelphia, PA, USA) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher) and 0.5 M EDTA (Thermo Fisher). Protein concentrations were quantified using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8–15%) and transferred onto polyvinylidene fluoride membranes (Merck Millipore). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 for 1 h at approximately 22–25 °C, followed by overnight incubation with primary antibodies at 4 °C. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoblot signals were detected using the Clarity Western ECL Substrate (Bio-Rad) and visualized using ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).
The following antibodies were used in this study: Anti-cleaved/pro-caspase (CASP)-9 (sc-56076), anti-cleaved/pro-CASP3 (#9662/9661), anti-Bcl2 (#509), anti-CDK4 (#601), anti-CDK6 (sc-7961), anti-cyclin E (sc-481), anti-NRF2 (sc-365949), anti-GPX4 (sc-166570), anti-glyceraldehyde 3-phosphate dehydrogenase (sc-365062), anti-DRP1 (sc-101270), anti-OPA1 (sc-393296), and anti-VDAC1 (sc-390996) antibodies (all from Santa Cruz), anti-E-cadherin (610181) and anti-N-cadherin (610920) antibodies (all from BD Biosciences), anti-cyclin D1 (2922S), anti-Bax (2772T), anti-LC3B (2775S), anti-PUMA (4976S), and anti-parkin (4211S) antibodies (all from Cell Signaling Technology), anti-SLC5A3 (ab110368), anti-ribonucleotide reductase catalytic subunit M1 (RRM1; ab137114), anti-Ki67 (ab15580), and anti-BrdU (ab6326) antibodies (all from Abcam), horseradish peroxidase-conjugated goat anti-mouse (7076S) and goat anti-rabbit (7074S) secondary antibodies (all from Cell Signaling Technology), Alexa Fluor 488 goat anti-mouse IgG (H + L) (A-11001), Cy5 goat anti-rabbit IgG (H + L) (A-10523), and Alexa Fluor 488 goat anti-rat IgG (H + L) (A-11006) secondary antibodies (all from Invitrogen).