Psat1

Antibodies obtained were directed against the following: EGFR (Cell Signaling, #2232), p-EGFR Tyr1068 (Cell Signaling, #2236); EGFR/EGFRvIII cocktail antibody (Novocastra); PARP (Cell Signaling, #9542), cleaved PARP (Cell Signaling #5625); PSAT1 (Abcam ab154055); SHMT1 (Cell Signaling, #80715); SHMT2 (Cell Signaling, #12762); MTHFD1 (Abcam ab70203); MTHFD2 (Abcam ab151447); MTHFD2 (Abcam ab56772); LC3A (Cell Signaling, #4599); LC3B (Cell Signaling, #3868); and β-actin (Ambion). Reagents used are Chloroquine (CQ, Sigma) and Nicotinamide Adenine Dinucleotide reduced form (NADH, Nacalai Tesque).

The total proteins of cells were extracted using RIPA buffer (Solarbio) containing protease inhibitor (Solarbio) and phosphatase inhibitor (Bosterbio, Wuhan, China). The cytoplasmic and nuclear proteins of cells were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Bosterbio). The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with nonfat milk and incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. Finally, the signals on the membranes were detected by enhanced chemiluminescence reagent under an Amersham Imager 600 (General Electric Company, USA), and the grey values of the protein bands were analysed by ImageJ software.
Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); Histone-H3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).