(A) Graphic comparison of PLK1 consensus substrate sequence with CTCF D220-F228. Green S/T with yellow encircled P, phosphorylation site at position 0. Red D/E, aspartic or glutamic acid. Blue Φ, hydrophobic amino acid. (B) Amino acid sequence identity heat map for the conserved kinases PLK1, ATM, and GSK3B. Nephrozoa species aligned in Figure 1F shown. (C) CTCF, CTCF Ser224-P, PCNA, and H3S10ph immunofluorescence performed on MEFs treated with DMSO or BI 6727 at the indicated concentrations for 12 hr. Nuclei counterstained with DAPI. Bar, 20 μm. % of n cells labeled with CTCF, CTCF Ser224-P, PCNA, or H3S10ph antibodies indicated. (D) PLK1 in vitro kinase assay with CTCF or dephosphorylated Casein substrates. Red *, phosphorylated CTCF. Red **, autophosphorylated PLK1. Red ***, phosphorylated casein. (E) In vitro kinase assay performed in parallel to (D without radioactive isotope. SDS-PAGE gel Coomassie stained. Red box, CTCF band excised for mass spectrometry analysis. (F) Manually validated mass spectra of CTCF peptide Tyr214-Lys244 with y and b ions identified. Phosphorylation event at Ser224 indicated by red s. (G) Immunofluorescence performed on TST-1 mESC metaphase chromosomes with the indicated antibodies. DNA stained with DAPI. (H) CTCF Ser224-P and H3K27me3 co-stain from (G) deconvolved.