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. 2020 Aug 6;11(8):649. doi: 10.1038/s41419-020-02825-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Western blot detection of acetylation levels of ectopically expressed MTHFD2(left) and endogenous MTHFD2 (right) after treated with 5 μm NAM for the duration indicated. Flag-MTHFD2 was immunoprecipitated from cell lysate and its acetylation was examined with a pan-acetylated lysine antibody (α-AcK). Endogenous MTHFD2 acetylation was examined with pan-acetylated lysine antibody (α-AcK) and site-specific K88 acetylation antibody (Ac-K88). Relative MTHFD2 acetylation was normalized by Flag protein or endogenous MTHFD2. b In vitro MTHFD2 acetylation assay. MTHFD2 proteins were incubated with different concentrations of acetyl-CoA as indicated. Protein acetylation level was analyzed, and relative MTHFD2 acetylation was normalized by His protein. c Identification of acetylated MTHFD2 peptides by tandem mass spectrum. Identified sites were shown in chat. d Acetylated MTHFD2 K88 was identified by a tandem mass spectrum. The identified peptide is shown. e K88 in MTHFD2 is evolutionarily conserved. The sequences around MTHFD2 K88 from different species were aligned. f K88 is the major acetylation residue of MTHFD2. Flag-tagged WT MTHFD2 or mutants (K44Q, K50Q, K88Q, and K104Q) were expressed in HEK293T cells, followed by treatments with or without 5 μm NAM. Flag-MTHFD2 was immunoprecipitated and its acetylation was examined with α-AcK. g Characterization of anti-acetyl-MTHFD2 (K88) (α-acK88) antibody. Specificity of antibody against acetylated K88 residue of MTHFD2 was determined by dot blot assay. Nitrocellulose membrane was spotted with different amounts of acetyl-K88 peptide or unmodified peptide and immunoblotted with anti-acetyl-MTHFD2 (K88) antibody. h Characterization of acetyl-MTHFD2 (K88) antibody. Acetylation level of MTHFD2-Flag, MTHFD2-K88R-Flag, or MTHFD2 K88Q-Flag ectopically expressed in HCT116 cells was measured by the site-specific K88 acetylation antibody (Ac-K88).