Protein Extraction and Immunoblotting

Cells were lysed with NP-40 buffer (1% NP40, pH 7.6, 150 mM NACL, 50 mM Tris, 1 mM Sodium orthovanadate, 1x Complete-EDTA, 0.2% Lauryl Maltoside) on ice for 30 min followed by centrifuging at 14,000 RPM at 4 °C for 20 min and protein content was determined using DC^TM Protein Assay kit according to the manufacturer’s protocol. Proteins were immunoblotted as described before (23 (link)). Briefly, 20 µg protein were denatured at 95 °C for 5 min in Laemmli buffer, separated on a gradient polyacrylamide gel (4–15%) (BioRad) and transferred onto a nitrocellulose membrane with the Trans-Blot Turbo system (BioRad) according to manufacturer’s guideline. Membranes were blocked in 5% milk for 1 h following primary antibody exposure over night at 4 °C. The following antibodies and related secondary antibodies (DAKO) were use with a dilution of 1:1,000 in TBST for Anti-PGDH3 (#HPA021241, Sigma), Anti-SHMT2 (#HPA020549, Atlas Antibodies), Anti-MTHFD1 (#HPA001290, Atlas Antibodies), TYMS (#AB232021, Abcam) and Anti-MTHFD2 (#H00010797-M01, Abnova) were used. Anti-PARK7 (1:1,000, #AB76008, Abcam) was used as loading control (24 (link)).