pvdf transfer membrane with low background fluorescence  (Millipore)

Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A p14 ARF structural features, including PSI-PRED4.0 secondary structure prediction (2 o Struc.), CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.). The CIDER analysis for p14 ARF ΔH1-3 is shown below. B Zoomed in regions from confocal fluorescence micrographs of NPM1-AF488 in condensates with p14 ARF (top) and p14 ARF ΔH1-3 (bottom). Scale bars = 10 µm. C Index of dispersion for NPM1 in condensates with p14 ARF (gray boxes, whiskers and trace; derived from n = 6, 6, 6, 7, 6, 6, 7, 6, 6, 6, 7 images) and p14 ARF ΔH1-3 (blue boxes and whiskers and trace, where n = 5, 4, 6, 6, 8, 6, 6, 6, 6, 6, 6 images). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The black arrow highlights the increased NPM1 saturation concentration, ΔC sat , upon substitution of p14 ARF hydrophobic residues to Gly/Ser spacer residues. The gray arrow highlights the reentrant phase transition, which occurs at elevated p14 ARF concentrations. D ΔG tr for NPM1 in condensates with p14 ARF (gray boxes, whiskers and trace, where n = 696, 42, 61, 159, 225, 285, 333, 276, 306, 227, 773 condensates) and p14 ARF ΔH1-3 (blue boxes, whiskers and trace, where n = 2561, 1787, 29, 31, 82, 92, 134, 153, 166, 145, 162 condensates). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The C sat for NPM1 increases when p14 ARF hydrophobic residues are substituted. The gray arrow highlights the destabilization of NPM1 during the reentrant phase transition. E CV-SANS curves for p14 ARF ΔH1-3-[ 2 H]-NPM1 condensates collected at 50% D 2 O, where p14 ARF ΔH1-3 is contrast matched ([ 2 H]-NPM1 detected; green trace), at 85% D 2 O where [ 2 H]-NPM1 is contrast matched (p14 ARF ΔH1-3 detected; blue trace), and p14 ARF ΔH1-3-NPM1 condensate at 100% D 2 O for full scattering intensity (NPM1 and p14 ARF ΔH1-3 detected; gray trace). All curves are offset for clarity. Scatter points represent the average, the error bars represent the uncertainty derived from the counting statistics of the SANS instrument, as described and cited in the Methods. F Schematic describing condensed NPM1 with extended IDR conformations. G Schematic describing condensed p14 ARF ΔH1-3 in an extended conformation. H FRAP of NPM1-AF488 within condensates shows that substitution of p14 ARF hydrophobic residues to Gly/Ser spacer residues restores NPM1 mobility. Scale bars = 1 µm. I FRAP recovery curves for p14 ARF -NPM1 and p14 ARF ΔH1-3-NPM1 condensates with fits, as described in Methods (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 curves for each condition, the p -value is shown in the figure). J NPM1-AF488 D App values extracted from the FRAP recovery curves in panel I (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 D App values for each condition, the p -value is shown in the figure).

Article Snippet: Gel electrophoresis was performed using 25–40 μg protein extracted from TRIzol cell lysates or equal volumes of protein extracts from sucrose gradient fractions in NuPAGE mini protein gels (Invitrogen), transferred for 1.5 h at 30 volt to PVDF Transfer Membrane with low background fluorescence (Millipore).

Techniques: Cider Assay, Residue, Fluorescence, Dispersion, Derivative Assay, Concentration Assay, Sublimation

Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A Zoomed in regions from fluorescence microscopy images of live DLD-1 NPM1-G (clone B11) cells, before and after 48 h of doxycycline induced p14 ARF -iRFP expression. Scale bars = 2 µm. B Z-score analysis of NPM1-GFP and p14 ARF -iRFP levels in DLD-1 NPM1-G cells, showing that p14 ARF and NPM1 levels are anti-correlated (statistical significance was assessed by two-sided Mann–Whitney U-test, n = 2272, 122, 54 cells, p -values are shown in the figure) C FRAP curves with fits, as described in Methods, for cells sorted from the DLD-1 NPM1-G population shown in B. The curves on the left are from a cell expressing a high level of nucleolar NPM1 (clone F6; green trace) and a low level of nucleolar p14 ARF (clone F6; blue trace). The curves on the right are from a cell expressing a low level of nucleolar NPM1 (clone G2; green trace) and a high level of nucleolar p14 ARF (clone G2; blue traces). In unsorted DLD-1 NPM1-G cells, D The D App and E mobility for nucleolar NPM1-GFP and p14 ARF -iRFP (small green and blue transparent markers, respectively, n = 45 cells) are reduced as nucleolar p14 ARF -iRFP levels increase. Reductions also occur as the duration of p14 ARF -iRFP expression is extended (large opaque markers; scatter points represent the mean and error bars represent the standard deviation, where n = 20 cells). F A schematic describing the correlated reductions in p14 ARF and NPM1 dynamics and their assembly into large molecular weight complexes within the granular component (GC) of the nucleolus.

Article Snippet: Gel electrophoresis was performed using 25–40 μg protein extracted from TRIzol cell lysates or equal volumes of protein extracts from sucrose gradient fractions in NuPAGE mini protein gels (Invitrogen), transferred for 1.5 h at 30 volt to PVDF Transfer Membrane with low background fluorescence (Millipore).

Techniques: Fluorescence, Microscopy, Expressing, MANN-WHITNEY, Standard Deviation, Molecular Weight