Mat2a

Rat lungs were homogenized, and cells were lysed in lysis buffer (50 mmol/L Tris–HCl pH 7.4, 150 mmol/l NaCl, 0.5% nonidet P-40) containing EDTA-free complete protease and phosphatase inhibitor (Sigma–Aldrich/Roche). Protein levels were measured using Bradford assays (Bio-Rad), after which 35 μg samples were run on SDS-PAGE gels, transferred to nitrocellulose membranes, blocked with either 5% bovine serum albumin or 4% milk, and subsequently incubated with primary and secondary antibodies and detected using SuperSignal West Pico Chemiluminescent Substrate (catalog no.: # 34080, Thermo Fisher Scientific) on iBright. Protein levels were determined through densitometric analysis using ImageJ software (NIH). The following previously validated primary antibodies were used: DNMT1 (Cell Signaling), DNMT3A (Cell Signaling), DNMT3B (Epigentek), MAT2A (Novus Biologicals), SAHH (Santacruz), NOS3 (Novus Biologicals), SOD2 (Novus Biologicals), SERPINE1 (Novus Biologicals), and ABRA (Novus biologicals). ACTB (Cell Signaling) was used as a loading control.

HUVECs were lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4, 1% IGEPAL, 0.1% sodium deoxycholate, 1 mM EDTA) supplemented with protease inhibitors (cOmplete ULTRA Mini, Roche) and PMSF. Proteins were separated using SDS-PAGE on precast TGX gradient gels (Biorad) and then transferred to polyvinylidine fluoride membranes using the Transblot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% BSA for 1 h, probed with primary antibodies overnight at 4 °C and then with peroxidase-conjugated secondary antibodies. The following antibodies were used: PKM1 (Cell Signaling Technology, #7067, 1:1000), PKM2 (Cell Signaling Technology, #4053, 1:1000), P21 (Cell Signaling Technology, #2947, 1:1000), P53 (Cell Signaling Technology, #9282, 1:500), P53 (Santa Cruz, sc-126, 1:1000), RELB (Cell Signaling Technology, #10544, 1:1000), MAT2A (Novus Biologicals, #NB110-94158, 1:2000), β-actin (Cell Signaling Technology, #8457, 1:1000), GAPDH (Cell Signaling Technology, #2118, 1:2000) and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2004, 1:10,000). For analysis, membranes were incubated with ECL (Clarity Western ECL Substrate, Biorad) and imaged using a ChemiDoc MP system (Biorad). Uncropped images of blots are presented in Supplementary Figure 8.

For MAT2A protein quantification, cells were harvested and lysed using RIPA buffer (1 % NP40, 0.5 % DOC, 0.1 % SDS, 50 mM TRIS pH 8.0, 80 mM NaCl, 50 mM NaF, 20 mM Na₄P₂O₇, 1 mM EDTA, 1 mM EGTA, and 1 Complete tablets Mini Easypack (Roche) for 10 mL). An equal protein amount was loaded on a 10% SDS gel with Laemmli buffer and PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific), and blotted on an Immobilon-FL PVDF membrane (Merck-Millipore). After blocking, primary antibodies (GAPDH, clone 14C10 from Cell Signaling, Danvers, MA, USA and MAT2A, polyclonal from Novus Biologicals, Centennial, CO, USA) and the secondary antibody (anti-rabbit IgG, IRDye 680RD, polyclonal from Li-Cor Biosciences, Lincoln, NE, USA) were used. Analysis was carried out at the Li-Cor Odyssey CLx using the Image Studio Software (both Li-Cor Biosciences) for documentation. Relative quantification was performed using glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) as the reference protein.