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. 2020 Dec 8;11:6297. doi: 10.1038/s41467-020-20080-9

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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 CARM1-interacting proteins were purified from HEK293T cells stably expressing Flag-CARM1 by co-immunoprecipitation with Flag-M2 agarose. Mass spectrometric analysis identified Pontin as a CARM1-interacting protein under glucose starvation. b The binding of Pontin to CARM1 was detected from the eluate of affinity chromatography by immunoblot analysis. c GST-pulldown assay for binding of CARM1 to Pontin and Reptin. d In vitro methylation assay using purified His-Pontin was performed with either HA-CARM1 WT or enzymatic activity-deficient R169A mutant. Immunoblotting with anti-asymmetric dimethyl arginine (Rme2a) antibody was performed after Ni-NTA pulldown. e In vitro methylation assay using purified GST-Pontin WT, R333A, R339A, or R333/339A mutant with HA-CARM1. Immunoblot analysis with anti-Rme2a antibody was performed after GST pulldown. f In vitro methylation assay of Pontin WT, R333K/R339K mutant, or R333A/R339A mutant proteins was performed with CARM1 proteins. g Ribbon diagram of Pontin (PDB 2C9O) showing its structure with R333 and R339 residues. h Sequence alignment of human Pontin with selected homologs. Conserved arginine residues (R333/R339) are highlighted with red. i The specificity of antibodies against methylated Pontin at R333/R339 residues was assessed by dot blot analysis. Methyl peptide sequence: IVIFASNR(me2)GNCVIR(me2)GTEDITS; Non-methyl peptide sequence: IVIFASNRGNCVIRGTEDITS. j In vitro methylation assay using purified GST-Pontin WT, R333A, R339A, or R333/339A with CARM1. Immunoblot analysis with anti-methyl-Pontin antibody was performed after GST pulldown. k In vitro methylation assay using purified GST-Pontin WT, R333K, R339K, or R333/339K proteins with CARM1. Immunoblot analysis with anti-methyl-Pontin and anti-Rme2a antibody was performed following GST pulldown. l In vitro methylation assay using purified His-Pontin with HA-CARM1 WT or R169A. Immunoblot analysis with anti-methyl-Pontin antibody was performed after Ni-NTA pulldown. Source data are provided as a Source Data file.