# anti phospho histone h2a x ser139 mouse monoclonal ab does not react to s139a in human pluripotent stem cells (Millipore)

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## Structured Review

Millipore
anti phospho histone h2a x ser139 mouse monoclonal ab does not react to s139a in human pluripotent stem cells

Anti Phospho Histone H2a X Ser139 Mouse Monoclonal Ab Does Not React To S139a In Human Pluripotent Stem Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/anti phospho histone h2a x ser139 mouse monoclonal ab does not react to s139a in human pluripotent stem cells/product/Millipore

Average 86 stars, based on 1 article reviews

Price from $9.99 to $1999.99

Anti Phospho Histone H2a X Ser139 Mouse Monoclonal Ab Does Not React To S139a In Human Pluripotent Stem Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/anti phospho histone h2a x ser139 mouse monoclonal ab does not react to s139a in human pluripotent stem cells/product/Millipore

Average 86 stars, based on 1 article reviews

Price from $9.99 to $1999.99

anti phospho histone h2a x ser139 mouse monoclonal ab does not react to s139a in human pluripotent stem cells - by Bioz Stars,
2024-10

86/100 stars

## Images

### 1) Product Images from "BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks"

**Article Title: **BRCA1 and 53BP1 Regulate Reprogramming Efficiency by Mediating DNA Repair Pathway Choice at Replication-associated Double-Strand Breaks

**Journal: **Cell reports

**doi: **10.1016/j.celrep.2024.114006

**Figure Legend Snippet:**A) Illustration of the BRCA1 polypeptide and a simplified interaction engagements. The C-terminal BRCT domain of BRCA1 interacts in a mutually exclusive manner with the phosphorylated isoforms of ABRAXAS, BACH1, or CtIP to form distinct BRCA1 complexes. In addition, BRCA1 harbors a coiled-coil motif that mediates its interaction with PALB2 and the recruitment of BRCA2 and RAD51 to sites of DNA damage. The mutant mouse alleles used in this study: Brca1tr encodes a pathogenic truncating mutation, denoted with a red arrow, that eliminates the BRCT domains. AbraxasS404A, Bach1S994A and CtipS326A each encode a missense mutation that eliminates a phosphorylation site necessary for the interaction of its protein product with the BRCT domain of BRCA1. B) Morphology and size of E13.5 embryos. The difference between wt ctrl and AABBCC was evaluated with an unpaired two-tailed student’s t-test.; wt ctrl n = 9, AABB n = 5, BBCC n = 4, AACC n = 3, AABBCC n = 3. C) Rad51 foci immunofluorescence and quantification in induced pluripotent stem (iPS) cell lines treated with 10Gy IR. Data was analyzed by one-way ANOVA.; wt ctrl n = 3, AABB n =3, BBCC n = 3, AACC n = 4, AABBCC n = 4; A = Abraxas S404A/S404A, B = Bach1 S994A/S994A, C = CtIP S326A/S326A; scale bar = 10μm. D) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cell lines, shown as a ratio of dual allele targeting in each genotype vs. control. Statistical analysis with one-way ANOVA, except for BBCC vs. AABBCC and AACC vs AABBCC, which used unpaired two-tailed student’s t-tests.; wt ctrl n = 4, AABB n = 4, BBCC n = 3, AACC n = 5, AABBCC n= 5. E) DNA fork stalling in immortalized MEFs. At least 150 DNA fibers were measured per genotype. Analysis with one-way ANOVA. F) Immunofluorescence and quantification for phospho-H2AX (S139). Foci were counted on reprogramming day 5 in > 138 cells/genotype. Statistical analysis with one-way ANOVA. Size bar = 5μm, applicable to all panels. G) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Analysis by one-way ANOVA from biological replicates. wt ctrl n = 3, AABB n = 4, BBCC n = 4, AACC n = 3, AABBCC n = 4.

**Techniques Used: **Mutagenesis, Two Tailed Test, Immunofluorescence, CRISPR, Control, Staining

**Figure Legend Snippet:**Related to Fig. S1. A) A schematic of Bard1-mediated SFP. The Bard1K607A point mutation prevents the recruitment of the Brca1/Bard1 heterodimer to reversed stalled replication forks, which makes them vulnerable to Mre11-dependent degradation. B) Immunofluorescence and quantification for double strand break (DSB) marker phospho - H2AX (S139). Foci were counted on reprogramming day 5 in ≥260 cells/genotype, statistical analysis with one-way ANOVA. scale bar = 5μm. C) Immunofluorescence and quantification of phospho -RPA(S33) on reprogramming day 5. Data from ≥240 cells/genotype and analyzed by one-way ANOVA.; The white arrows point to foci. scale bar = 5μm. D) Cell proliferation plots on reprogramming day 5. Arrested cells retain CFSE and are detectable as a bright peak by flow cytometry. Analysis by one-way ANOVA; wt ctrl n = 3, Brca1tr/+ n = 2, Bard1K607A/K607A n = 3. E) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Analysis with one-way ANOVA.; wt ctrl n = 6, Brca1tr/+ n = 6, Bard1K607A/+ n = 4, Bard1K607A/K607A n = 4, n=biological replicates.

**Techniques Used: **Mutagenesis, Immunofluorescence, Marker, Flow Cytometry, Staining

**Figure Legend Snippet:**Related to Fig. S2 A) A schematic for rescuing SFP in BRCA1 mutant cells by ablation of Smarcal1. B) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype. Analysis by one-way ANOVA. C) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype relative to control. Statistical analysis with one-way ANOVA; wt ctrl n = 9, Brca1tr/+ n = 3, Brca1tr/tr n = 3, Brca1tr/tr Smarcal1−/− n = 3. D) Immunofluorescence and quantification for double strand break (DSB) marker phospho-H2AX (S139). Foci were counted on reprogramming day 5 (≥410 cells/genotype) and statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho-RPA(S33) on reprogramming day 5. Data collected from ≥140 cells per genotype and analyzed by one-way ANOVA. F) Cell proliferation analysis with CFSE on reprogramming day 5. Statistical analysis with one-way ANOVA.; wt ctrl n = 4, Smarcal1+/− n = 4, Smarcal1−/− n = 5, Brca1tr/tr n = 2, Brca1tr/tr Smarcal1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5. Analysis by one-way ANOVA.; wt ctrl n = 3, Smarcal1+/− n = 3, Smarcal1−/− n = 3, Brca1tr/tr n = 3, Brca1tr/tr Smarcal1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. The number of AP positive colonies is shown as a ratio to wild type. Data analysis with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, Smarcal1+/− n = 4, Smarcal1−/− n = 2, Brca1tr/tr n = 12, Brca1tr/tr Smarcal1+/− n = 4, Brca1tr/tr Smarcal1−/− n = 4. n= biological replicates.

**Techniques Used: **Mutagenesis, CRISPR, Control, Immunofluorescence, Marker, Staining

**Figure Legend Snippet:**Related to Fig. S3. A) A schematic for rescuing HDR in Brca1 mutant cells by ablation of 53bp1. Relevant substrates that can be repaired by HDR or NHEJ are shown. B) CRISPR/Cas9 based HDR assay with induced pluripotent stem (iPS) cells lines. Data is shown as a ratio of dual allele targeting in each genotype to dual allele targeting in the control. Statistical analysis using one-way ANOVA, except for the comparison between wt ctrl and 53bp1−/−, evaluated with a two-tailed, unpaired student’s t-test.; wt ctrl n = 9, Brca1tr/+ n = 3, 53bp1−/− n = 5, Brca1tr/tr n = 3, Brca1tr/tr53bp1+/− n = 2, Brca1tr/tr53bp1−/− n = 4. C) DNA fiber analysis in a fork stalling assay with hydroxyurea (HU) on reprogramming day 5. At least 120 DNA fibers were measured per genotype and data was analyzed by one-way ANOVA. D) Immunofluorescence and quantification for phospho-H2AX (S139). Foci counted on reprogramming day 5 (≥410 cells/genotype), statistical analysis performed with one-way ANOVA. E) Immunofluorescence and quantification of ssDNA marker phospho RPA(S33) on reprogramming day 5. ≥140 cells per genotype, analyzed by one-way ANOVA. For control and BRCA1, images are identical for Fig. 3 and Fig. 4 for panels c and d. F) Cell proliferation analysis with CFSE dye on reprogramming day 5. Statistics with one-way ANOVA.; wt ctrl n = 4,, 53bp1’/- n = 5, 53bp1−/− n = 3, Brca1tr/tr n = 2, Brca1tr/trSmarcal1−/− n = 3, Brca1tr/tr 53bp1−/− n = 3. G) Apoptosis analysis with Annexin V and propidium iodide (PI) on reprogramming day 5, analyzed by one-way ANOVA.; wt ctrl n = 3, 53bp1+/− n = 3, 53bp1−/− n = 2, Brca1tr/tr n = 3, Brca1tr/tr 53bp1−/− n = 3. H) Alkaline phosphatase (AP) staining and reprogramming efficiency quantification. Number of AP positive colonies is shown as a ratio to wild type, analyzed with one-way ANOVA.; wt ctrl n = 7, Brca1tr/+ n = 6, 53bp1+/− n = 7, 53bp1−/− n = 8, Brca1tr/tr n = 12, Brca1tr/tr 53bp1−/− n = 7. I) Quantification of Nanog positive colonies in the indicated genotypes, analyzed with one-way ANOVA.; n = 3 for each genotype. J) AP staining and reprogramming efficiency quantification in human 1023 fibroblasts from adult skin biopsy in control and 53BP1 knockdown (KD) conditions. Cells were fixed on day 25 post reprogramming factor transduction, statistical analysis using an unpaired, two-tailed student’s t-test.; ctrl n = 16, 53BP1 KD n = 16. Samples with n=2 are not used for statistical comparisons. All numbers indicated are biological replicates.

**Techniques Used: **Mutagenesis, CRISPR, Control, Comparison, Two Tailed Test, Immunofluorescence, Marker, Staining, Knockdown, Transduction

**Figure Legend Snippet:**KEY RESOURCES TABLE

**Techniques Used: **Purification, Transduction, Recombinant, Knock-Out, Plasmid Preparation, Software, Imaging