Fig. 3

PADI4 citrullinates HP1γ in vitro. a Recombinant GST-HP1γWT, R38K, R39K or R38/9K mutant proteins were incubated with recombinant GST-PADI4 in the presence or absence of activating calcium as indicated. Reactions were resolved by SDS-PAGE and analysed by immunoblot analysis using an anti-peptidyl-citrulline antibody. Ponceau S staining of the transferred proteins serves as a loading control (lower panel). Bands highlighted by the red boxes were quantified using ImageJ. Total intensities after background correction are indicated (right panel). Statistical analysis was performed using two-way ANOVA multiple corrections with Sidak post hoc test. Bars represent ± SD, n = 3 (**p values from left to right: 0.002, 0.003, 0.008) (Additional file 4: Figure S3A). b GST-HP1γWT or R38/9K mutant proteins were incubated with GST-PADI4, with or without calcium, in the presence or absence (w/o) of unmethylated H3(1–16) peptides or H3K9me3(1–16) peptides, as indicated. Ponceau S staining of the transferred proteins serves as a loading control (lower panel). Bands highlighted by the red boxes were quantified using ImageJ and background corrected. Increased citrullination of HP1γWT in the presence of H3K9me3 peptides compared to unmethylated H3 peptide is displayed as fold change intensity (right panel). Statistical analysis was performed using unpaired Student’s t test. Bars represent ± SD, n = 3 (**p value: 0.004) (Additional file 4: Figure S3B)