MOUSE ANTI PCNA VMA00018 from Bio-Rad (Formerly AbD Serotec)

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Cell reports
December 10 2019

Fig 1: Recruitment of CPD to Telomeres Lacking TRF2 and POT1a/b-TPP1(A) Co-localization of p-RPA32, RAD51, and Flag-ATRIP with telomeres (arrowheads) in Nbs1+/+, Nbs1−/− MEFs, or NBS-ILB1 cells. Telomeres were visualized by peptide nucleic acid-fluorescent in situ hybridization (PNA-FISH) (red), indicated proteins (green), and nuclei (blue). Scale bar for all panels: 5 μm.(B) Percentage of cells containing ≥5 TIFs in (A). Data are the mean of 2 independent experiments ± SD; n > 200 nuclei analyzed per experiment. ****p < 0.0001 by 1-way ANOVA. ns, non-significant.(C) Co-IP of HA-Claspin with GFP-DONSON or Myc-PCNA in 293T cells. Input, 5% of the total cell lysate. γ-Tubulin, loading control.(D–F) Localization of Claspin (D), PCNA (E), and DONSON (F) in Nbs1−/− MEFs expressing indicated DNAs. Endogenous DONSON TIFs were detectable only in HA-claspin-expressing cells. Telomeres were visualized by PNA-FISH (red), and endogenous CPD was visualized with the indicated antibodies (green). Scale bar for all panels: 5 μm.(G) Percentage of cells containing ≥5 CPD+ foci co-localizing with telomeres, detected in (D)–(F).(H) Co-expression of HA-Claspin with GFP-DONSON (top panel) or HA-Claspin, Myc-PCNA, and GFP-DONSON (bottom panel) in U2OS cells. Top panel: GFP-DONSON (green) and HA-Claspin(red). Bottom panel: Myc-PCNA (red) and GFP-DONSON (green). For bothpanels, anti-TRF2 antibody visualized telomeres (white).(I) HA-Claspin and GFP-DONSON co-localized with BrdU+ foci in U2OS cells. GFP-DONSON (green), BrdU (red), and DAPI (blue). Scale bar for all panels: 5 μm.
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Cell reports
December 10 2019

Fig 2: Genomic Instability in Tumors Derived from MMTV-Cre; p53F/F; Pot1aF/F Mice(A) CPD, SMARCAL1, p-CHK1, and p-RPA32 expression in MMTV-Cre; p53Δ/Δ; Pot1aΔ/Δ breast tumors (#1, 3, and 4), sarcoma (#2), and salivary gland tumor (#5). Tumor #6 is MMTV-Cre; p53Δ/Δ; Pot1aF/ΔΔ. γ-Tubulin, loading control. Bottom panel: PCR genotyping.(B) Detection of Claspin and PCNA (green) and telomeres (red) in tumor #1.(C) Percentage of cells containing ≥3 Claspin/PCNA (CP+) TIFs in (B). Scale bar for all panels: 5 μm.(D) γ-H2AX TIFs in tumor cell lines. Telomere PNA-FISH (red), antibody staining (green), and nuclei (blue). Scale bar for all panels: 5 μm.(E) Percentage of cells containing ≥5 γ-H2AX TIFs in (D). Data represent the mean of 2 independent experiments ± SD; n > 200 nuclei analyzed per experiment. *p = 0.01, **p = 0.001, ***p = 0.0001 by 1-way ANOVA.(F) Percentage of cells containing ≥3 p-RPA32 TIFs in tumor cell lines.(G) SMARCAL1 TIFs in tumor cell lines.(H) Interstitial telomeres (red arrow), chromosome and sister chromatid fusions (orange arrow) in Pot1a deleted mouse tumor cell lines. TelG-FAM, TelC-Cy3, and DAPI were used to visualize fused chromosomes. Scale bar for all panels: 5 μm.(I) Quantification of the number of interstitial telomeres and chromosome fusions in (H). Data represent the average of 2 independent experiments ± SD from a minimum of 70 metaphases.(J) Spearman’s correlation between claspin and POT1 gene expression in colon adenocarcinoma obtained from TCGA data.(K) Schematic depicting CPD functions. See text for details.
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Cell reports
December 10 2019

Fig 3: CPD Promotes A-NHEJ-Mediated Chromosome Fusions in the Absence of NBS1(A) Percentage of cells containing =5 ?-H2AX+ TIFs in Nbs1+/+ and Nbs1-/- MEFs expressing the indicated DNAs. Data are the mean of 3 independent experiments ± SD; n > 300 nuclei analyzed per experiment. ***p = 0.0002, ****p < 0.0001 by 1-way ANOVA.(B) Telomere fusion frequencies in Nbs1+/+ cells treated with shTrf2, TPP1?RD, or both. Data are the average of 3 independent experiments ± SD from a minimum of 140 metaphases. *p = 0.01, **p = 0.001, ****p < 0.0001 by 1-way ANOVA. ns, non-significant.(C) Telomere fusion frequencies in Nbs1-/- treated with shTrf2, TPP1?RD, or both. Data are the average of 3 independent experiments ± SD from a minimum of 140 metaphases. **p = 0.001, ***p = 0.0001, ****p < 0.0001 by 1-way ANOVA. ns, non-significant.(D) Domain organization of Claspin. Replication fork-interacting domain, basic patch I, basic patch II, PCNA-interacting domain, CHK1-binding domain, and C-terminal acidic patch. The red dashed line separates ClaspinN-Ter (amino acids [aa] 1–679) and ClaspinC-Ter (aa 679–1,332).(E) Nbs1-/- MEFs expressed the indicated DNA constructs for 120 h. TelG-FAM, TelC-Cy3, and DAPI-labeled metaphase spreads to visualize fused chromosomes (arrowheads). Scale bar for all panels: 5 µm.(F) Percentage of cells containing =5 ?-H2AX TIFs observed in (E). Data are the mean of 2 independent experiments ± SD; n > 200 nuclei analyzed per experiment. **p = 0.01, ***p = 0.001, by 1-way ANOVA. ns, non-significant.(G) Telomere fusion frequencies in (E). Data are the average of 2 independent experiments ± SD from a minimum of 70 metaphases. **p = 0.001, ***p = 0.0001, ****p < 0.0001 by 1-way ANOVA. ns, non-significant.(H) Co-IP of HA-ClaspinWT, HA-ClaspinN-Ter, or HA-ClaspinC-Ter with GFP-DONSON in 293T cells. Input, 5% of the total cell lysate. ?-Tubulin, loading control.(I) U2OS cells transiently transfected with HA-ClaspinWT, HA-Claspin3A, HA-ClaspinN-Ter, or HA-ClaspinC-Ter with GFP-DONSON for 48 h. GFP-DONSON (green), HA-Claspin (red), and DAPI to visualize nuclei (blue). Scale bar for all panels: 5 µm.(J) Percentage of cells containing =5 ?-H2AX+ TIFs in Nbs1-/- MEFs infected with indicated DNA constructs. Data are the mean of 2 independent experiments ± SD; n > 200 nuclei analyzed per experiment. **p = 0.001, ***p = 0.0003 by 1-way ANOVA. ns, non-significant.(K) Telomere fusion frequencies. Data are the average of 3 independent experiments ± SD from a minimum of 120 metaphases. ***p = 0.001, ****p < 0.0001 by 1-way ANOVA. ns, non-significant.

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The MOUSE ANTI PCNA Antibody from Bio-Rad (Formerly AbD Serotec) is a Mouse Monoclonal antibody to PCNA, and proliferating cell nuclear antigen. This antibody recognizes Homo Sapiens, Human, Mouse, Rat, and Non-Human Primate antigen. The MOUSE ANTI PCNA Antibody has been validated for the following applications: Immunoprecipitation, and Western Blot.

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MOUSE ANTI PCNA from Bio-Rad (Formerly AbD Serotec)

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MOUSE ANTI PCNA from Bio-Rad (Formerly AbD Serotec)

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