Mthfd2

The BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used to detect the concentration of protein extracted from cultured cells or resected tumors. Equivalent protein samples were separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride (PVDF; Amersham Pharmacia, Little Chalfont, UK). The membrane was incubated with the primary antibodies for MTHFD2 (Cell Signaling Technology, Danvers, MA, USA), Ki-67 (Cell Signaling Technology), Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2 (Santa Cruz Biotechnology), CytC (Santa Cruz Biotechnology), or β-actin (Cell Signaling Technology), followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). The blots were detected using an enhanced chemiluminescence kit (ECL; Amersham Biosciences, Piscataway, NJ, USA) and visualized with the Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).

Antibodies against Flag (Cell Signaling Technology, Cat#2368), pan-acetylated lysine (Cell Signaling Technology, Cat#9441), MTHFD2 (Cell Signaling Technology, Cat#41377), SIRT4 (Abclonal, Cat#A7585), CUL3 (Abcam, Cat#ab75851), β-actin (AOGMA, Cat#9601), and HA (SAB, Cat#T501) were purchased. To generate a site-specific antibody against acetyl K50 of MTHFD2 [acMTHFD2 (K50)], the synthesized peptide (GL Biochem) was coupled to keyhole limpet hemocyanin to immunize rabbits and the post-immunization serum was collected.