Extended Data Fig. 4: Mutant HMGB1 protein and synthetic IDR peptide.
From: Aberrant phase separation and nucleolar dysfunction in rare genetic diseases
(a) Chromatograms from size-exclusion chromatography (SEC) of wild type and mutant mEGFP-HMGB1 fusion proteins. Selected fractions highlighted with red. (b) SDS-PAGE analysis of purified proteins after His-Tag column purification before SEC and after SEC purification steps. Analysis was performed once for each protein prep. (c) SDS-PAGE of SEC purified HMGB1 proteins followed by immunoblotting with anti-EGFP and anti-HMGB1 antibodies. Analysis was performed once for each protein prep. (d) Representative images from droplet formation assays performed with 5’FAM-labeled HMGB1-IDR variant peptides at indicated concentrations in the presence of 20 ng/µl RNA or without RNA. Experiment was replicated 2 times with similar results. (e) Quantification of the relative amount of condensed protein of 5’FAM-HMGB1-IDR peptides at the indicated concentrations. Data displayed as mean ± SD from 5 image fields examined per condition. (f) Representative images from droplet formation assays performed with 2.5 µM 5’FAM-labeled HMGB1-IDR synthetic peptides at indicated RNA concentrations. Experiment was replicated 2 times with similar results. (g) Quantification of the relative amount of condensed protein of 2.5 µM 5’FAM-HMGB1-IDR peptides at the indicated RNA concentrations. Data displayed as mean ± SD from 5 image fields examined per condition. (h) Scheme of co-droplet assays. (i) (top) Representative images of droplets formed by 5’FAM-HMGB1-IDR peptide mixed with pre-assembled mCherry-labeled MED1-IDR, HP1α or NPM1 droplets. (bottom) Example images from 5 µM 5’FAM-HMGB1 mutant peptide mixed with mCherry labeled NPM1 droplets with uneven distribution of the peptide within droplet. (j) Dual fluorescence plot quantification of 5’FAM and mCherry fluorescence intensities in mCherry-labeled NPM1, MED1-IDR or HP1α droplets mixed with synthetic HMGB1 IDR peptides. Each dot represents one droplet, and the size of the dot is proportional to the size of the droplet. Scale bars, 5 µm (d,f) or 1 µm (g,i).
