p-s10 histone h3 antibody  (Jackson Immuno)

Journal: bioRxiv

Article Title: Potent synthetic lethality between PLK1 and EYA-family inhibitors in tumours of the central and peripheral nervous system

doi: 10.1101/2025.01.19.633804

Figure Lengend Snippet: (A) Representative immunofluorescence images showing G2 and M-phase cells stained with p-S46 TCTP, p-S10 H3 and DAPI. (B) Representative quantitative image-based cytometry plot showing G2 and M-phase cell cycle gates as determined by staining intensity of p-S10 H3 and DAPI. (C) Cellular quantitation of p-S46 TCTP intensity in G2-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.01, **** p ≤ 0.0001). (D) Cellular quantitation of p-S46 TCTP intensity in M-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). (E) Representative immunofluorescence images showing an S-phase cell stained with RAD51, EdU, and DAPI. Individual RAD51 foci are indicated with red outline. (F) Representative quantitative image-based cytometry plot showing S-phase cell cycle gate as determined by staining intensity of EdU and DAPI. (G) Cellular quantitation of RAD51 foci number in S-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.01). (H) Quantitation of RAD51 foci intensity in S-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001).

Article Snippet: Cells were incubated with anti-p-S46 TCTP (Cell Signalling 5251) and anti-p-S10 H3 primary antibodies (Cell Signalling 9701) overnight at 4 ° C followed by 3 x 10 minute washes in PBS.

Techniques: Immunofluorescence, Staining, Cytometry, Quantitation Assay, Comparison

Journal: Cell reports

Article Title: FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination

doi: 10.1016/j.celrep.2023.113523

Figure Lengend Snippet: (A) Representative images of phospho-H3 S10-stained nuclei (blue), EdU foci (green), and FANCD2 (red) foci from APH-treated RPE-1 wild-type and PCNA K164R cell lines. EdU foci almost exclusively (>95%) colocalized with FANCD2 foci. Scale bar, 5 μm. (B) FANCD2 foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNA K164R (maroon), RAD18 −/− (salmon), and RAD18 −/− ;PCNA K164R (pink) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ***p < 0.001. FANCD2 foci overlapped with EdU foci in 98% of nuclei. (C) EdU foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNA K164R (maroon), RAD18 −/− (salmon), and RAD18 −/− ;PCNA K164R (pink) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ***p < 0.001. (D) FANCD2 foci quantification from two biological replicates in 293T wild-type (blue) and PCNA K164R cells (maroon) treated with 450 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by one-way ANOVA with Tukey’s multiple comparison test with ***p < 0.001. (E) EdU foci quantification from two biological replicates in 293T wild-type (blue) and PCNA K164R cells (maroon) treated with 450 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by one-way ANOVA with Tukey’s multiple comparison test with ***p < 0.001. (F) Western blot analyses of chromatin associated PCNA, with or without 40 J/m 2 UV treatment, with histone H2AX as the loading control from RPE-1 wildtype, PCNA K164R , and reverted PCNA K164 cells. (G) FANCD2 foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNA K164R (maroon), and reverted PCNA K164 (purple) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ****p < 0.001. (H) EdU foci quantification from two biological replicates in RPE-1 wild-type (blue), PCNA K164R (maroon), and reverted PCNA K164 (purple) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ****p < 0.001.

Article Snippet: p -Histon H3 (S10) , Active Motif , 61623 RRID:AB_2793707.

Techniques: Staining, Comparison, Western Blot, Control

Journal: Cell reports

Article Title: FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination

doi: 10.1016/j.celrep.2023.113523

Figure Lengend Snippet: (A) Representative images of phospho-H3 S10-stained nuclei (blue), EdU foci (green), and PLA foci (red) from APH-treated RPE-1 cells. Scale bar, 5 μm. (B) PLA foci quantification from at least three biological replicates in RPE-1 wild-type (blue), FANCD2 −/− (green), PCNA K164R (maroon), and reverted PCNA K164 (purple) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. Significance was calculated by Kruskal-Wallis with Dunn’s multiple comparison test with ****p > 0.001. (C) Quantification of overlapping PLA and EdU foci from at least three biological replicates in RPE-1 wild-type (blue), FANCD2 −/− (green), PCNA K164R (maroon), and reverted PCNA K164 (purple) cells treated with 300 nM APH. Number (n) of nuclei quantified is listed. (D) In wild-type (PCNA K164 ) cells (left), PCNA is ubiquitinated at K164 in response to replication stress by RAD18. During S phase, this facilitates the switch from processive DNA polymerase to TLS polymerases (for example, Pol η) to complete DNA synthesis. At alternative loci, ubiquitinated PCNA promotes the ubiquitination and chromatin association of FANCD2. During G2/M phase, PCNA recruits subunits of TLS Pol ζ to FANCD2-marked foci to initiate MiDAS and then facilitates processive replication through association with components of Pol δ. In mutant (PCNA K164R ) cells (right), PCNA ubiquitination is impaired. Subsequently, TLS polymerase and FANCD2 recruitment to stressed replication forks is reduced in S and G2/M phases, causing the persistence of under-replicated genomic loci.

Article Snippet: p -Histon H3 (S10) , Active Motif , 61623 RRID:AB_2793707.

Techniques: Staining, Comparison, DNA Synthesis, Ubiquitin Proteomics, Mutagenesis

Journal: Cell reports

Article Title: FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination

doi: 10.1016/j.celrep.2023.113523

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: p -Histon H3 (S10) , Active Motif , 61623 RRID:AB_2793707.

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, In Situ, Sequencing, Software, Imaging, Flow Cytometry, Transfection, Microscopy