Fig. 2: SIRT3 is the major deacetylase for MTHFD2.

a, b SIRT3 interacts MTHFD2. Interactions between MTHFD2 and SIRT3 were determined when proteins were co-expressed with each other in HEK293T cells. c, d endogenous SIRT3 interacts with MTHFD2 in vivo. Whole-cell lysates were immunoprecipitated with control IgG, anti-SIRT3, or anti-MTHFD2 antibody, and the precipitated proteins were detected with anti-MTHFD2 or anti-SIRT3 antibody, respectively. e SIRT3 deacetylates MTHFD2 in a dose-dependent manner. Acetylation levels were detected of ectopically expressed MTHFD2(left) and endogenous MTHFD2 (right) after transfected with plasmids that express increasing amounts of SIRT3. Whole-cell lysates were immunoprecipitated with Flag-beads(left) or anti-MTHFD2 antibody(right), and the precipitated proteins were detected by an acetylation assay. f SIRT3-knockout increases MTHFD2 acetylation level. SIRT3-knockout generated by CRISPR/Cas9 were analyzed by western blotting for SIRT3 expression. Whole-cell lysates of HCT116 WT cells or SIRT3-knockout cells were immunoprecipitated with anti-MTHFD2 antibody, and the precipitated proteins were detected by an acetylation assay. g Acetylation levels were detected of ectopically expressed(left) and endogenous MTHFD2 (right) after transfected with empty vector or with SIRT3(WT) or SIRT3-H248Y (H248Y). Whole-cell lysates were immunoprecipitated with Flag-beads(left) or anti-MTHFD2 antibody(right), and the precipitated proteins were detected by an acetylation assay. h SIRT3 deacetylates MTHFD2 in vitro. Recombinant human (rh) SIRT3 deacetylates the site-specific K88-acetylated MTHFD2 proteins incubated with NAD+ or NAM, and acetylation level was determined by Ac-K88 and pan-AcK. i Recombinant catalytically inactive (rh) SIRT3 mutant H248Y and the site-specific K88-acetylated MTHFD2 incubated with NAD+ or NAM, and acetylation level was determined by Ac-K88 and pan-AcK.