Fig. 2

Efficient binding of HP1γ to H3K9me3 requires R38 and R39 in vitro. a Coomassie-blue stained gel showing the results from a pulldown assay analysing binding of recombinant GST-HP1γWT and GST-HP1γR38/9A proteins to H3(1–16) peptides or H3K9me3(1–16) peptides, as indicated. 25% of input amounts are shown. Binding intensities of gel bands from multiple experiments (Additional file 2: Figure S2A) were quantified using ImageJ and were normalised to total inputs (right hand graph). Statistical analysis was performed using unpaired, 2 tailed Student’s t-test. Bars represent ± SD, n = 3 (*p value: 0.021, **p value: 0.008, ns.: not significant, p value: 0.181). The sequences of the peptides used are indicated at the top. b Real-time binding kinetics of GST-HP1γ proteins using bio-layer interferometry (BLI). BLI sensorgrams are shown profiling the normalised binding of recombinant GST-HP1γWT and GST-HP1γR38/9A to H3(1–16) peptides (H3: right panel) and H3K9me3(1–16) peptides (H3K9me3: left panel), as indicated. Association (30–150 s) and dissociation (150–270 s) were each measured over the course of 120 s. The protein concentrations used were WT: 28.0 μM; R38/9A: 28.3 μM. Results of one experiment repeated at a range of different protein concentrations are shown (Additional file 2: Figure S2C