Protease and phosphatase inhibitors

The cells were seeded in 60-mm dishes at a density of 7 × 10⁵ cells per dish and transfected with indicated plasmids (5 μg) for 24 h. Briefly, the cells were homogenized in 0.5 mL of homogenization buffer (10 mM HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 5 mM sodium EDTA, 5 mM sodium EGTA, and 250 mM sucrose) supplemented with protease and phosphatase inhibitors (MedChemExpress), then centrifuged at 1000× g at 4 °C for 7 min. The pellet, containing the crude nuclear fraction, was discarded, and the supernatant was centrifuged at 12,000× g at 4 °C for 15 min. The resulting supernatant comprised the cytosol, and the pellet containing the membrane fraction was dissolved in lysis buffer (10 mM Tris HCl pH 6.8, 100 mM NaCl, 1% SDS, 1 mM EDTA, and 1 mM EGTA) supplemented with protease and phosphatase inhibitors. The whole cell lysate, cytosolic, and membrane samples were mixed individually with 4 × SDS loading buffer, boiled at 95 °C for 5 min, and subjected to SDS-PAGE and immunoblotting analysis.

Brain tissue was micropunched for CeA. The tissue was transferred to an RIPA protein extraction buffer (Beyotime Biotechnology, China) containing protease and phosphatase inhibitors (MedChemExpress, China), homogenized, and centrifuged at 12,000 g at 4°C for 15 min. The supernatant was separated, and protein concentration was measured using the BCA assay (Beyotime Biotechnology, China). Western blot was carried with the previously described methodology. Briefly, samples were separated using SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore), which were then blocked with 5% non-fat milk in TBST for 1 h. The membranes were then incubated with primary antibodies against Nat8l/ASPA/GCP II/GCP III/NAAGSI and β-actin overnight at 4°C. After washing for three times in TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibodies in TBST for 1 h at room temperature and then visualized with an ECL luminometer. Quantitative analysis was performed with ImageJ software. All experiments were performed at least three times.

Cells were lysed with RIPA lysis buffer (Solarbio, China) containing protease and phosphatase inhibitors (MedChemExpress, United States). An equal amount of protein in each sample was isolated by SDS‒PAGE electrophoresis, transferred to a PVDF membrane (Millipore, United States), and then blocked with 5% fat-free milk or 3% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h. The membrane was then incubated with the primary antibody, and the general Western blotting assay procedure was followed. For immunoprecipitation, the starting steps were as described above but involved the use of IP lysate (Thermo Fisher Scientific, United States). A portion of the protein solution was taken to directly detect the corresponding protein expression, while the rest of the solution was added to the primary antibody and magnetic beads (Bimake, United States), respectively, according to the manufacturer’s instructions. The beads were washed with wash buffer (Thermo Fisher Scientific, United States) and heated to 100 °C for 5 min using protein loading buffer (ABclonal, China). Immunoblotting was then performed as described above.

Proteins were extracted by a radioimmunoprecipitation lysis buffer (VWR Chemicals, Radnor, PA, USA) with protease and phosphatase inhibitors (MedChem Express, Monmouth Junction, NJ, USA). After centrifugation at 13,000 rpm for 10 min at 4 °C, the supernatant was collected to determine protein concentrations using the bicinchoninic acid (BCA) assay (Visual Protein, Taipei, Taiwan). Samples containing 30 μg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Biomate, Taipei, Taiwan). The membranes were blocked with 10% skim milk in TBS-T (20 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and then incubated with primary antibodies, including antibodies against E-cadherin (IReal Biotechnology, Hsinchu, Taiwan), N-cadherin (IReal Biotechnology), vimentin (IReal Biotechnology), Snail (IReal Biotechnology), GPX4 (Proteintech, Chicago, IL, USA), and β-actin (Merck Millipore) at 4 °C overnight. The blots were then washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The blots were detected by enhanced chemiluminescence (Millipore), and the relative signal intensity was quantified using the ImageJ software (U.S. National Institute of Health, Bethesda, MD, USA).

Proteins were isolated using Cell Culture Lysis 1 × Reagent (Promega) containing a mixture of protease and phosphatase inhibitors (MedChemExpress). Protein signals on membranes were developed using electrochemiluminescence (ECL) Western blot analysis substrates (Daeillab Service) and analysed. Details are summarized in Appendix S1.

Lung tissues or cells were fully lysed in RIPA buffer (Beyotime, China) containing a fresh mixture of protease and phosphatase inhibitors (MedChemExpress, United States).
The entire process was performed at 4°C. After centrifugation at 12000 r/min for 20 min, the supernatant was added to 5× protein sample loading buffers (Epizyme, China) and boiled for 10 min. A BCA protein kit (Thermo, USA) was used to determine protein concentrations. Denatured proteins were separated by 10% SDS-PAGE (Epizyme, China) and then transferred onto methanol-activated PVDF membranes (Millipore, USA) at a constant current of 400 mA. After blocking with 5% skim milk for 1 h, membranes were incubated with different primary antibodies overnight at 4°C. After washing the PVDF membrane several times, it was incubated with the appropriate secondary antibody (1:2,000) for 1 h at room temperature. Subsequently, ECL (GE Healthcare, United Kingdom) was used to visualize protein expression, and ImageJ software was used to analyze the band intensities.