Fig. 6

Interaction of the Pgm CRD with histone H3. a In vitro pulldown assay of P. tetraurelia histones with GST or GST-Pgm(692–768) (GST-CRD). b In vitro pulldown assay of P. tetraurelia histones with MBP or MBP-Pgm(692–768) (MBP-CRD). MBP fusion proteins and histone H3 were revealed on western blots using α-MBP-HRP and α-H3 antibodies, respectively (see Fig. S4 for the control of histone preparations and full-size blots with molecular weight markers). c Amino acid composition of the synthetic peptides used in this study. Methylated lysines are underlined. Hydrophobic residues are in red, residues with positively charged side chains in blue, residues with uncharged polar side chains in purple. C-terminally biotinylated peptides (−b) used in ELISA assays are indicated. d ELISA assays with 100 nM MBP or MBP-Pgm(692–768) (MBP-CRD) against unmodified H3(1–19), trimethylated H3(1–19)K4me3 and H3(1–19)K9m3, control peptides Scrambled 1 (*: TECAN absorbance detection at 450 nm close to saturation) and Scrambled 2, unmodified H3(16–35) and trimethylated H3(16–35)K27me3. p-values were calculated using the Wilcoxon-Mann-Whitney test calculator (https://ccb-compute2.cs.uni-saarland.de/wtest/); sample sizes m = 3, n = 3 and test variant H1: (H3(1–19)K4me3 signal) < (H3(1–19) signal) and H1: (H3(1–19)K4me3 signal) < (H3(1–19)K9me3 signal). e ELISA assays with MBP alone or MBP-Pgm(692–768) (MBP-CRD) against the H3(1–19) peptide. One hundred nM of each MBP fusion protein was loaded into H3(1–19)-coated wells either in absence (left panel) or in presence of 10 mM EDTA (right panel). Error bars represent the standard deviation between technical triplicates