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Addgene inc pines80 addgene plasmid
Pines80 Addgene Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna5+frt+to/pm41875887-830-33-34?v=Addgene+inc
Average 92 stars, based on 9 article reviews

pines80 addgene plasmid - by Bioz Stars, 2026-08

92/100 stars

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Image Search Results

(a) Graph plotting the predicted disorder score of human 53BP1 amino acid sequence, as calculated by IUPred2A. Scores above 0.5 (threshold marked by the horizontal black line) indicate predicted IDRs. Full-length protein was used as input. Horizontal blue lines mark regions of the protein which remain disordered upon binding (based on FuzPred predictions). (b) Schematic representation of full-length 53BP1 highlighting annotated protein domains (top), and the fragments designed for this study (bottom, from F1 to F12). OD = oligomerization domain, GAR = glycine-arginine-rich motif, Tud = Tudor domain, UDR = ubiquitination-dependent recruitment motif, BRCT = BRCA1 C-terminal domain. (c) Graphs plotting the probabilities of residues to undergo disorder-to-order transition upon binding (p DO ) for individual 53BP1 fragments, as computed by FuzPred. Scores higher than 0.5 (threshold marked by the horizontal black line) indicate transitions to ordered states upon binding. Horizontal blue lines mark regions of the fragment which remain disordered upon binding. (d) Predicted secondary structure of the 53BP1 F6 fragment as computed by PSIPRED. Disordered residues are outlined in blue. Structured residues are colored in yellow (strand) and pink (helix).

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Graph plotting the predicted disorder score of human 53BP1 amino acid sequence, as calculated by IUPred2A. Scores above 0.5 (threshold marked by the horizontal black line) indicate predicted IDRs. Full-length protein was used as input. Horizontal blue lines mark regions of the protein which remain disordered upon binding (based on FuzPred predictions). (b) Schematic representation of full-length 53BP1 highlighting annotated protein domains (top), and the fragments designed for this study (bottom, from F1 to F12). OD = oligomerization domain, GAR = glycine-arginine-rich motif, Tud = Tudor domain, UDR = ubiquitination-dependent recruitment motif, BRCT = BRCA1 C-terminal domain. (c) Graphs plotting the probabilities of residues to undergo disorder-to-order transition upon binding (p DO ) for individual 53BP1 fragments, as computed by FuzPred. Scores higher than 0.5 (threshold marked by the horizontal black line) indicate transitions to ordered states upon binding. Horizontal blue lines mark regions of the fragment which remain disordered upon binding. (d) Predicted secondary structure of the 53BP1 F6 fragment as computed by PSIPRED. Disordered residues are outlined in blue. Structured residues are colored in yellow (strand) and pink (helix).

Article Snippet: For the expression of 53BP1 fragments as recombinant proteins, 53BP1 fragments were amplified from the plasmid encoding FL 53BP1 (pcDNA5-FRT/TO-eGFP-53BP1, 60813, Addgene) using different primer pairs [FF1/Rev361 for 53BP1 F1 (residues 1-361); FF302/Rev718 for 53BP1 F2 (residues 302-718); FF667/Rev1052 for 53BP1 F3 (residues 667-1052); FF1231/Rev1483 for 53BP1 F6 (residues 1231-1483); FF1053/Rev1711 for 53BP1 F11 (residues 1053-1711); FF1711/Rev1972 for 53BP1 F12 (residues 1711-1972)] and Phusion DNA polymerase (NEB).

Techniques: Sequencing, Binding Assay, Ubiquitin Proteomics

(a) Graph plotting the probability of disorder-to-order transitions upon binding (p DO ) for full-length 53BP1, as computed by FuzPred. Scores above 0.5 (threshold marked by the horizontal black line) indicate transitions to ordered states upon binding. (b) Summary table of FuzPred and FuzDrop results for 53BP1 fragments. Colored cells indicate values exceeding the defined threshold for each parameter. The probability to form disordered interactions (p DD ) is defined as the median of the residue-based values of the fragment as computed by the FuzPred method . Fragments with dominantly disordered interactions (p DD > 0.5) are colored in blue. DPRs were defined as stretches of at least 25 consecutive residues with p DP ≥ 0.60 as computed by the FuzDrop method . The number (n DPR ) and the average length of the droplet-promoting regions (L DPR ) are displayed. F1-F3 are equipped with multiple, shorter droplet-promoting regions, while F11 with multiple, longer DPRs. All DPRs exhibit disordered interactions (p DD (DPR) > 0.5). The MBM was used to assess the propensity of DPRs to convert into a more solid-like state . DPRs defined as context-dependent (MBM ≥ 0.59) are colored in green.

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Graph plotting the probability of disorder-to-order transitions upon binding (p DO ) for full-length 53BP1, as computed by FuzPred. Scores above 0.5 (threshold marked by the horizontal black line) indicate transitions to ordered states upon binding. (b) Summary table of FuzPred and FuzDrop results for 53BP1 fragments. Colored cells indicate values exceeding the defined threshold for each parameter. The probability to form disordered interactions (p DD ) is defined as the median of the residue-based values of the fragment as computed by the FuzPred method . Fragments with dominantly disordered interactions (p DD > 0.5) are colored in blue. DPRs were defined as stretches of at least 25 consecutive residues with p DP ≥ 0.60 as computed by the FuzDrop method . The number (n DPR ) and the average length of the droplet-promoting regions (L DPR ) are displayed. F1-F3 are equipped with multiple, shorter droplet-promoting regions, while F11 with multiple, longer DPRs. All DPRs exhibit disordered interactions (p DD (DPR) > 0.5). The MBM was used to assess the propensity of DPRs to convert into a more solid-like state . DPRs defined as context-dependent (MBM ≥ 0.59) are colored in green.

Techniques: Binding Assay, Residue

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) SDS-PAGE gel (Coomassie-stained) showing all the purified recombinant 53BP1 fragments used in this study, at their highest achievable purity. (b) Mass photometry analysis of purified 53BP1 F6 fragment confirms sample monodispersity and homogeneity. Raw data acquisition at 200 nM displayed on a ratiometric contrast scale (left). Mass distribution at 200 nM obtained after calibration using BSA and BAM standards (right). Measurements were performed in SEC buffer using a TwoMP Instrument (Refeyn). Each condition was tested in duplicate. (c-f) Thermal unfolding analysis of purified 53BP1 F6 fragment confirms protein stability. (c) nanoDSF F 350/330nm profile (top) and its first derivative (bottom). T m = 48.57 ± 0.76°C (dashed line). (d) Turbidity (backreflection) profile (top) and its first derivative (bottom). T agg = 76.48 ± 0.07 °C (dashed line). (e) Scattering profile (SLS). T onset = 43.50 ± 0.51 °C. (f) Cumulant Radius analysis of DLS data. T onset = 47.01 ± 0.24 °C. All the experiments were performed in triplicate at 2 mg/mL in SEC buffer, using a 25 to 95 °C temperature gradient with a ramp of 0.5 °C/min, on a Prometheus Panta NT.48 Instrument (NanoTemper GmBH).

Techniques: SDS Page, Staining, Purification, Recombinant, Nano Differential Scanning Fluorimetry

(a) Representative images of 53BP1 fragment droplets formation in presence of increasing amounts of RNA, with their relative quantification of droplets number per field and mean droplet area. Data are presented as mean ± SEM from n = 3 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “0 ng RNA” group (* P < 0.05, ** P < 0.01). Differential Interference Contrast (DIC) channel is shown; scale bar = 10 µm. (b) Representative images of F6 droplets dissolution over time after RNase A treatment, with their relative quantification. RNA was added to the F6 fragment, droplets were allowed to form and then incubated with RNase A or acetylated BSA as a control. The graph shows the total droplets area over time after treatment addition. Data are presented as mean ± SD from n = 5 independent experiments. Statistical analysis was performed at the endpoint using an unpaired t -test (** P < 0.01). DIC channel is shown; scale bar = 5 µm. (c) Representative images of F6 droplets formation in presence of 1000 ng RNA and 100 mM ammonium acetate (NH 4 OAc) or 5% 1,6-hexanediol. Data are presented as mean ± SEM from n = 4 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “RNA” group (* P < 0.05). DIC channel is shown; scale bar = 10 µm. (d) Representative images of F6 droplets formation in presence of increasing amounts of PAR, with their relative quantification of droplets number per field and mean droplet area. F6 with 1000 ng RNA was used as a positive control. Data are presented as mean ± SEM from n = 3 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “1000 ng RNA” group (**** P < 0.0001). DIC channel is shown; scale bar = 10 µm.

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Representative images of 53BP1 fragment droplets formation in presence of increasing amounts of RNA, with their relative quantification of droplets number per field and mean droplet area. Data are presented as mean ± SEM from n = 3 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “0 ng RNA” group (* P < 0.05, ** P < 0.01). Differential Interference Contrast (DIC) channel is shown; scale bar = 10 µm. (b) Representative images of F6 droplets dissolution over time after RNase A treatment, with their relative quantification. RNA was added to the F6 fragment, droplets were allowed to form and then incubated with RNase A or acetylated BSA as a control. The graph shows the total droplets area over time after treatment addition. Data are presented as mean ± SD from n = 5 independent experiments. Statistical analysis was performed at the endpoint using an unpaired t -test (** P < 0.01). DIC channel is shown; scale bar = 5 µm. (c) Representative images of F6 droplets formation in presence of 1000 ng RNA and 100 mM ammonium acetate (NH 4 OAc) or 5% 1,6-hexanediol. Data are presented as mean ± SEM from n = 4 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “RNA” group (* P < 0.05). DIC channel is shown; scale bar = 10 µm. (d) Representative images of F6 droplets formation in presence of increasing amounts of PAR, with their relative quantification of droplets number per field and mean droplet area. F6 with 1000 ng RNA was used as a positive control. Data are presented as mean ± SEM from n = 3 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “1000 ng RNA” group (**** P < 0.0001). DIC channel is shown; scale bar = 10 µm.

Techniques: Quantitative Proteomics, Dissolution, Incubation, Control, Positive Control

(a) Representative images of 53BP1 fragment droplets formation in presence or absence of 10% PEG. The experiment was performed in duplicate, with similar results. DIC channel is shown; scale bar = 10 µm. (b) Representative images of F6 droplets dissolution and reformation after RNase A treatment and inhibition, with their relative quantification of droplets number per field and mean droplet area. After droplets formation with RNA, RNase A was added to the well, and imaged 30 min post-treatment; the mixture was incubated with RNaseOUT for 15 min to inactivate RNase A, followed by addition of extra RNA and imaging 30 min later. Data are presented as mean ± SD from n = 1 independent experiments. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “RNA” group (* P < 0.05, **** P < 0.0001). DIC channel is shown; scale bar = 10 µm. (c) Representative images of F6 behavior in presence of increasing amounts of cellular RNA or genomic DNA. The experiment was performed in triplicate, with similar results. DIC channel is shown; scale bar = 10 µm.

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Representative images of 53BP1 fragment droplets formation in presence or absence of 10% PEG. The experiment was performed in duplicate, with similar results. DIC channel is shown; scale bar = 10 µm. (b) Representative images of F6 droplets dissolution and reformation after RNase A treatment and inhibition, with their relative quantification of droplets number per field and mean droplet area. After droplets formation with RNA, RNase A was added to the well, and imaged 30 min post-treatment; the mixture was incubated with RNaseOUT for 15 min to inactivate RNase A, followed by addition of extra RNA and imaging 30 min later. Data are presented as mean ± SD from n = 1 independent experiments. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test against “RNA” group (* P < 0.05, **** P < 0.0001). DIC channel is shown; scale bar = 10 µm. (c) Representative images of F6 behavior in presence of increasing amounts of cellular RNA or genomic DNA. The experiment was performed in triplicate, with similar results. DIC channel is shown; scale bar = 10 µm.

Techniques: Dissolution, Inhibition, Quantitative Proteomics, Incubation, Imaging

(a) (Left) 1 H- 15 N HSQC spectrum and backbone assignments of 53BP1 F6. (Right) 1 H- 15 N HSQC resonance intensities for the assigned residue positions (top); the gray bars indicate residues with missing resonances. NMR experimental secondary chemical shifts (middle) and NMR chemical shift-based secondary structure analysis (bottom) using the SSP program. HSQC spectra were collected on n > 3 independent 53BP1 F6 samples. (b) (Left) 1 H- 15 N HSQC overlay of 53BP1 F6 spectra with (red) and without (blue) the addition of Torula yeast RNA. (Right) Chemical shift perturbation (top) and peak intensity reduction (bottom) induced by the addition of Torula yeast RNA mapped to 53BP1 F6 sequence. Peak shifts observed upon adding RNA suggest nearby RNA interactions. Lysine and arginine residues are highlighted in pink and red, respectively; the gray bars indicate missing peaks. HSQC spectra were collected on n > 3 independent 53BP1 F6 samples.

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) (Left) 1 H- 15 N HSQC spectrum and backbone assignments of 53BP1 F6. (Right) 1 H- 15 N HSQC resonance intensities for the assigned residue positions (top); the gray bars indicate residues with missing resonances. NMR experimental secondary chemical shifts (middle) and NMR chemical shift-based secondary structure analysis (bottom) using the SSP program. HSQC spectra were collected on n > 3 independent 53BP1 F6 samples. (b) (Left) 1 H- 15 N HSQC overlay of 53BP1 F6 spectra with (red) and without (blue) the addition of Torula yeast RNA. (Right) Chemical shift perturbation (top) and peak intensity reduction (bottom) induced by the addition of Torula yeast RNA mapped to 53BP1 F6 sequence. Peak shifts observed upon adding RNA suggest nearby RNA interactions. Lysine and arginine residues are highlighted in pink and red, respectively; the gray bars indicate missing peaks. HSQC spectra were collected on n > 3 independent 53BP1 F6 samples.

Techniques: Residue, Sequencing

(a) (Top) Schematic representation of 53BP1 F6 fragment sequence, highlighting the OD and the GAR motif. (Bottom) Sequence alignment of the GAR motif region of 53BP1 orthologs from Saccharomyces cerevisiae (RAD9), Caenorhabditis elegans (hsr-9), Schizosaccharomyces pombe (crb2), Danio rerio , Xenopus tropicalis , Homo sapiens , Mus musculus , and Rattus norvegicus . (b) EMSA showing a titration of WT, R→A, R→K F6 fragments against a fixed concentration of 63-nt synthetic ssRNA (1 µM). Quantification was performed by calculating the ratio of bound RNA band intensity over total lane intensity for each condition. Data are presented as mean ± SD from n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test against “F6 WT” group at each concentration (** P < 0.01, **** P < 0.0001). (C) Representative images of WT, R→A, R→K F6 droplets formation in presence of increasing amounts of RNA, with their relative quantification of droplets number per field and mean droplet area. Data are presented as mean ± SEM from n = 3 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test against “F6 WT” group at each concentration (*** P < 0.001, **** P < 0.0001). DIC channel is shown; scale bar = 10 µm. (d) EMSA showing a titration of WT, R→K, R→A GAR peptides against a fixed concentration of 63-nt synthetic ssRNA (2 µM). Quantification was performed by calculating the ratio of bound RNA band intensity over total lane intensity for each condition. Data are presented as mean ± SD from n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test against “GAR WT” group art each concentration (** P < 0.01, **** P < 0.0001). Two-way ANOVA revealed a significant main effect of the protein variant (**** P < 0.0001) and RNA concentration (* P < 0.05), but no significant interaction between the two factors, indicating that the effect of protein variant on droplet formation was consistent across all RNA concentrations tested. (e) Binding curves between 21-nt ssRNA and WT, R→A, R→K GAR peptides, obtained by FP. Polarization values were plotted against peptide concentration, while RNA concentration was kept constant (50 nM). Data are presented as mean ± SD from n = 4 independent experiments. The data points were fitted by non-linear regression using a one-site specific binding model. The solid line represents the best-fit curve. The apparent dissociation constants (K d ) and maximum binding capacities (B max ) are listed in the table (± SEM; n.d. = not determinable under tested conditions). (f) (Left) Phase separation assay showing a titration of WT, R→A, R→K GAR peptides against RNA (1000 ng). The experiment was performed 4 times, with similar results. DIC channel is shown; scale bar = 10 µm. (Right) Graph showing peptide concentrations categorized as positive (filled circle) or negative (open circle) for aggregate-like structures, with half-and-half circles indicating variability in the presence of aggregate structures at that peptide concentration.

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) (Top) Schematic representation of 53BP1 F6 fragment sequence, highlighting the OD and the GAR motif. (Bottom) Sequence alignment of the GAR motif region of 53BP1 orthologs from Saccharomyces cerevisiae (RAD9), Caenorhabditis elegans (hsr-9), Schizosaccharomyces pombe (crb2), Danio rerio , Xenopus tropicalis , Homo sapiens , Mus musculus , and Rattus norvegicus . (b) EMSA showing a titration of WT, R→A, R→K F6 fragments against a fixed concentration of 63-nt synthetic ssRNA (1 µM). Quantification was performed by calculating the ratio of bound RNA band intensity over total lane intensity for each condition. Data are presented as mean ± SD from n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test against “F6 WT” group at each concentration (** P < 0.01, **** P < 0.0001). (C) Representative images of WT, R→A, R→K F6 droplets formation in presence of increasing amounts of RNA, with their relative quantification of droplets number per field and mean droplet area. Data are presented as mean ± SEM from n = 3 independent experiments. Each data point represents the mean of multiple fields from a single replicate. Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test against “F6 WT” group at each concentration (*** P < 0.001, **** P < 0.0001). DIC channel is shown; scale bar = 10 µm. (d) EMSA showing a titration of WT, R→K, R→A GAR peptides against a fixed concentration of 63-nt synthetic ssRNA (2 µM). Quantification was performed by calculating the ratio of bound RNA band intensity over total lane intensity for each condition. Data are presented as mean ± SD from n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test against “GAR WT” group art each concentration (** P < 0.01, **** P < 0.0001). Two-way ANOVA revealed a significant main effect of the protein variant (**** P < 0.0001) and RNA concentration (* P < 0.05), but no significant interaction between the two factors, indicating that the effect of protein variant on droplet formation was consistent across all RNA concentrations tested. (e) Binding curves between 21-nt ssRNA and WT, R→A, R→K GAR peptides, obtained by FP. Polarization values were plotted against peptide concentration, while RNA concentration was kept constant (50 nM). Data are presented as mean ± SD from n = 4 independent experiments. The data points were fitted by non-linear regression using a one-site specific binding model. The solid line represents the best-fit curve. The apparent dissociation constants (K d ) and maximum binding capacities (B max ) are listed in the table (± SEM; n.d. = not determinable under tested conditions). (f) (Left) Phase separation assay showing a titration of WT, R→A, R→K GAR peptides against RNA (1000 ng). The experiment was performed 4 times, with similar results. DIC channel is shown; scale bar = 10 µm. (Right) Graph showing peptide concentrations categorized as positive (filled circle) or negative (open circle) for aggregate-like structures, with half-and-half circles indicating variability in the presence of aggregate structures at that peptide concentration.

Techniques: Sequencing, Titration, Concentration Assay, Quantitative Proteomics, Variant Assay, Binding Assay

(a) Sequence of the synthetic GAR peptides used in this study, comprising 53BP1 GAR motif in wild-type form with arginines (GAR WT), or mutated to alanines (GAR R→A) or to lysines (GAR R→K). (b-c) Representative images of AlphaFold 3-predicted structure of WT, R→A and R→K F6 fragment (b) or GAR peptides (c) with 63-nt synthetic ssRNA. The stoichiometric ratio of protein or peptide to RNA is 1:1. The predictions were run 10 times for each condition with 10 autogenerated seeds, yielding similar results. The structure is colored by pLDDT. The GAR motif is colored magenta. In the middle, the predicted aligned error (PAE) plot. Black lines delineate borders between different entities. Blue boxes indicate regions corresponding to the GAR motif. On the right, a zoom-in of the simulated interaction between the GAR motif and RNA. Dashed lines represent putative hydrogen bonds (cyan), or cation-π interactions (orange).

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Sequence of the synthetic GAR peptides used in this study, comprising 53BP1 GAR motif in wild-type form with arginines (GAR WT), or mutated to alanines (GAR R→A) or to lysines (GAR R→K). (b-c) Representative images of AlphaFold 3-predicted structure of WT, R→A and R→K F6 fragment (b) or GAR peptides (c) with 63-nt synthetic ssRNA. The stoichiometric ratio of protein or peptide to RNA is 1:1. The predictions were run 10 times for each condition with 10 autogenerated seeds, yielding similar results. The structure is colored by pLDDT. The GAR motif is colored magenta. In the middle, the predicted aligned error (PAE) plot. Black lines delineate borders between different entities. Blue boxes indicate regions corresponding to the GAR motif. On the right, a zoom-in of the simulated interaction between the GAR motif and RNA. Dashed lines represent putative hydrogen bonds (cyan), or cation-π interactions (orange).

Techniques: Sequencing

(a-c) DDM analysis of F6 WT condensates. (a) Wavevector-dependent relaxation rate Γ ( q ) obtained from DDM analysis of a single F6 WT droplet. The dashed red line corresponds to the best fit to the rate with a linear model Γ ( q ) = v DDM q , from which v DDM = (32 ± 1) μm / s is estimated. Top left: ROI where DDM is applied. The ROI is chosen to be smaller than the probed F6 WT droplet to avoid edge effects. Scale bar = 5 µm. (b) Time evolution of capillary velocity and amplitude (inset) computed from DDM for a single F6 WT droplet. (c) Capillary velocity for different F6 WT droplets estimated on the day of sample preparation and on subsequent days. Data are presented as box plots. Statistical analysis was performed using Mann-Whitney U-test or Wilcoxon rank sum test (* P < 0.05). (d-f) In vitro FRAP analysis of F6 WT and F6 R→K condensates. (d) FRAP measurements for two representative F6 WT and F6 R→K mutant condensates, respectively. Scale bar = 2 µm. Right: azimuthally averaged normalized intensity profiles measured at the time after photobleaching for which images are shown. (e) Normalized integrated time-dependent concentration after photobleaching ⟨ C ∗ ⟩( t ) for F6 WT condensate (blue circles) and F6 R→K mutant condensate (red squares), respectively. Dashed lines correspond to the best-fitting curves based on the model described in . Vertical dashed lines indicate the time points reported in (d). (f) Effective diffusion coefficient D obtained by fitting 3D pure diffusion model with infinite boundary conditions to the normalized concentration for two different days after sample preparation. Data are presented as box plots. Statistical analysis was performed using Mann-Whitney U-test or Wilcoxon rank sum test (*** P < 0.001). (g-j) Coalescence analysis of F6 WT and F6 R→K condensates. (g) Representative images of F6 WT and F6 R→K mutant condensates undergoing coalescence in the stationary regime ( t ∼ 30 min) during time after contact at t = 0. Scale bar = 2 µm. On the right the aspect ratio AR as a function of time. The dashed lines correspond to the best fit to the aspect ratio with a simple exponential model AR ( t ) = ( AR 0 − AR ∞ ) exp(− t / τ ) + AR ∞ . (h) Coalescence velocity v 0 for different coalescence events observed in time after sample preparation for F6 WT condensate (blue circles) and F6 R→K mutant condensate (red squares), respectively. The white area marks the region t 0 > 1.6 × 10 G s for which the sample has reached steady-state. (i) Asymptotic aspect ratio AR ∞ at steady-state as a function of the condensate radius L . (j) Coalescence velocity v 0 for F6 WT (blue circles) and F6 R→K mutant (red squares) in the stationary state. Data are presented as box plots. Statistical analysis was performed using Mann-Whitney U-test or Wilcoxon rank sum test (*** P < 0.001). (k-l) FRAP analysis in irradiated BJ cells expressing a mCherry-tagged 53BP1 WT construct, performed at different time points after DNA damage. Cells were irradiated with 20 Gy, and FRAP was performed at 2 h, 8 h, 24 h, 1 week post-irradiation. (k) Fluorescence recovery time course after FRAP on BJ mCherry-53BP1 cells. Curves have been normalized to a 0 (intensity at the bleaching frame) to 1 (pre-bleaching intensity) scale for easier comparison. Data are presented as mean ± SEM from n = 3 independent experiments. (l) FRAP half-recovery times ( t 1/2 ) on BJ mCherry-53BP1 cells, derived from the curves shown in (k). Data are presented as box plots from 3 independent experiments (16-39 cells analyzed per time point per experiment), with different shapes indicating the different replicates. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test (** P < 0.01, **** P < 0.0001).

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a-c) DDM analysis of F6 WT condensates. (a) Wavevector-dependent relaxation rate Γ ( q ) obtained from DDM analysis of a single F6 WT droplet. The dashed red line corresponds to the best fit to the rate with a linear model Γ ( q ) = v DDM q , from which v DDM = (32 ± 1) μm / s is estimated. Top left: ROI where DDM is applied. The ROI is chosen to be smaller than the probed F6 WT droplet to avoid edge effects. Scale bar = 5 µm. (b) Time evolution of capillary velocity and amplitude (inset) computed from DDM for a single F6 WT droplet. (c) Capillary velocity for different F6 WT droplets estimated on the day of sample preparation and on subsequent days. Data are presented as box plots. Statistical analysis was performed using Mann-Whitney U-test or Wilcoxon rank sum test (* P < 0.05). (d-f) In vitro FRAP analysis of F6 WT and F6 R→K condensates. (d) FRAP measurements for two representative F6 WT and F6 R→K mutant condensates, respectively. Scale bar = 2 µm. Right: azimuthally averaged normalized intensity profiles measured at the time after photobleaching for which images are shown. (e) Normalized integrated time-dependent concentration after photobleaching ⟨ C ∗ ⟩( t ) for F6 WT condensate (blue circles) and F6 R→K mutant condensate (red squares), respectively. Dashed lines correspond to the best-fitting curves based on the model described in . Vertical dashed lines indicate the time points reported in (d). (f) Effective diffusion coefficient D obtained by fitting 3D pure diffusion model with infinite boundary conditions to the normalized concentration for two different days after sample preparation. Data are presented as box plots. Statistical analysis was performed using Mann-Whitney U-test or Wilcoxon rank sum test (*** P < 0.001). (g-j) Coalescence analysis of F6 WT and F6 R→K condensates. (g) Representative images of F6 WT and F6 R→K mutant condensates undergoing coalescence in the stationary regime ( t ∼ 30 min) during time after contact at t = 0. Scale bar = 2 µm. On the right the aspect ratio AR as a function of time. The dashed lines correspond to the best fit to the aspect ratio with a simple exponential model AR ( t ) = ( AR 0 − AR ∞ ) exp(− t / τ ) + AR ∞ . (h) Coalescence velocity v 0 for different coalescence events observed in time after sample preparation for F6 WT condensate (blue circles) and F6 R→K mutant condensate (red squares), respectively. The white area marks the region t 0 > 1.6 × 10 G s for which the sample has reached steady-state. (i) Asymptotic aspect ratio AR ∞ at steady-state as a function of the condensate radius L . (j) Coalescence velocity v 0 for F6 WT (blue circles) and F6 R→K mutant (red squares) in the stationary state. Data are presented as box plots. Statistical analysis was performed using Mann-Whitney U-test or Wilcoxon rank sum test (*** P < 0.001). (k-l) FRAP analysis in irradiated BJ cells expressing a mCherry-tagged 53BP1 WT construct, performed at different time points after DNA damage. Cells were irradiated with 20 Gy, and FRAP was performed at 2 h, 8 h, 24 h, 1 week post-irradiation. (k) Fluorescence recovery time course after FRAP on BJ mCherry-53BP1 cells. Curves have been normalized to a 0 (intensity at the bleaching frame) to 1 (pre-bleaching intensity) scale for easier comparison. Data are presented as mean ± SEM from n = 3 independent experiments. (l) FRAP half-recovery times ( t 1/2 ) on BJ mCherry-53BP1 cells, derived from the curves shown in (k). Data are presented as box plots from 3 independent experiments (16-39 cells analyzed per time point per experiment), with different shapes indicating the different replicates. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test (** P < 0.01, **** P < 0.0001).

Techniques: Sample Prep, MANN-WHITNEY, In Vitro, Mutagenesis, Concentration Assay, Diffusion-based Assay, Irradiation, Expressing, Construct, Fluorescence, Comparison, Derivative Assay

(a) Immunoblot for 53BP1, TRF2 and actin (loading control) in Trf2 F/F 53bp1 −/− MEFs expressing empty vector (EV), full-length wild-type 53BP1 (WT) or its GAR mutants (R→A or R→K), before or 96 h after Cre-mediated Trf2 deletion with Hit&Run Cre. (b) Representative metaphase spreads in the indicated MEFs at 96 h after Cre-mediated Trf2 deletion. Telomeres were detected with Alexa Fluor 488-OO-(TTAGGG) 3 (green), DNA with DAPI (magenta). Arrows indicate telomere fusions. The boxed regions are enlarged in the right row. Scale bar = 10 µm. (c-d) Quantification of telomere fusions shown in (b) at 96 h (c) or 120 h (d) after Cre-mediated Trf2 deletion. Data are presented as median from 3 independent experiments (10 metaphases each). Each data point represents the percentage of telomeres fused in one metaphase, with different shapes indicating the different replicates. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Journal: bioRxiv

Article Title: The glycine-arginine-rich motif of 53BP1 modulates RNA interactions necessary for its liquid-liquid phase separation during DNA Damage Response

doi: 10.64898/2026.01.30.702603

Figure Lengend Snippet: (a) Immunoblot for 53BP1, TRF2 and actin (loading control) in Trf2 F/F 53bp1 −/− MEFs expressing empty vector (EV), full-length wild-type 53BP1 (WT) or its GAR mutants (R→A or R→K), before or 96 h after Cre-mediated Trf2 deletion with Hit&Run Cre. (b) Representative metaphase spreads in the indicated MEFs at 96 h after Cre-mediated Trf2 deletion. Telomeres were detected with Alexa Fluor 488-OO-(TTAGGG) 3 (green), DNA with DAPI (magenta). Arrows indicate telomere fusions. The boxed regions are enlarged in the right row. Scale bar = 10 µm. (c-d) Quantification of telomere fusions shown in (b) at 96 h (c) or 120 h (d) after Cre-mediated Trf2 deletion. Data are presented as median from 3 independent experiments (10 metaphases each). Each data point represents the percentage of telomeres fused in one metaphase, with different shapes indicating the different replicates. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Techniques: Western Blot, Control, Expressing, Plasmid Preparation

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