A Scatter plot comparing mean-fold change for CARM1-BirA* fusion vs. BirA* alone with abundance in published negative control AP-MS datasets (%CRAPome). Green dots represent proteins (i) with a cutoff frequency of ≥80% CRAPome and an average spectral count fold change ≥1.2 or (ii) with a cutoff frequency of <80% CRAPome but the average spectral count fold change ≥3.0. Known substrates of CARM1 are indicated as red, and E2F-targets, histone binding proteins, and MYC-targets are shown in yellow, blue, and violet, respectively. See also supplementary data 1 (HEL cells, n = 1) and 2 (K562 cells, n = 1). B Proteins were immunoprecipitated from HEL cell extracts that express MYC-tagged CARM1 (WT and non-phosphorylatable mutants), using an anti-MYC antibody; immunoblotting was then performed using an anti-MYC antibody and anti-RUNX1 mouse antibody. C Doxycycline-inducible short hairpin RNAs (shRNAs) directed against CARM1 decreased CARM1 protein levels and the ADMA levels of RUNX1-R223 and -R319 as well as well-established targets, such as PABP1-R455/R460 and BAF155-R1064. D Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 (Cas9)-mediated non-phosphorylatable CARM1 mutants decreased the ADMA levels of RUNX1-R223 and -R319 as well as PABP1 and BAF155. E Expression of total and asymmetry dimethylated RUNX1, PABP1, and BAF155 were assessed in HEL cells treated with RUX 250 nM or DMSO control for 5 days. Fresh media with RUX or DMSO was added on days 0, 2, and 4. Quantification of the ADMA levels of RUNX1, BAF155, and PABP1 at 5 days after RUX treatment are shown in the right panels. Data represent the mean ± SD. n = 3, one-way ANOVA. The relative cell viability at day 5 was 85% for cells treated with RUX, compared to those cultured with DMSO (P = 0.005)(n = 3, biological replicates). F The levels of ADMA RUNX1, BAF155, and PABP1 were measured in HEL cells treated with RUX (or DMSO) after EPZ025654 treatment for 5 days followed by a wash-out phase lasting up to 3 days (labeled as day 8). The relative cell viability at day 8 was 86.8% for cells treated with RUX compared to those cultured with DMSO (P = 0.006)(n = 3 biological replicates). The experiments were repeated at least two times independently. B, D
n = 2, (C) n = 3.