mouse α brca1  (Merck & Co)

Journal: bioRxiv

Article Title: DNA end-resection is stimulated by an interaction between BRCA1 exon 11 and TOPBP1

doi: 10.64898/2026.01.09.698382

Figure Lengend Snippet: (A) Lysates of RPE1 hTERT cell lines either TP53 -/- or TP53 -/- BRCA1 -/- virally reconstituted with a dox-inducible BRCA1-TY-1 cDNA (FL), BRCA1-TY-1 exon11-less (Δ11) cDNA or an empty vector (EV) control analyzed by western blotting to indicate BRCA1 variant expression 24 hr post dox induction (1 μg/mL). (B) TP53 -/- or TP53 -/- BRCA1 -/- virally reconstituted with dox-inducible FL BRCA1-TY-1 or BRCA1-TY-1-Δ11 were irradiated with 10 Gy and 4 hr post-IR analyzed by IF microscopy to detect p-RPA foci formation (n=3, mean + SEM, paired t-test, p-values are shown). A minimum of 100 cells were counted per replicate. (C) Same cells used in (A) and (B) were irradiated as in (B) and IF microscopy was performed to quantify RAD51 foci (n=2/3, mean + SEM, paired t-test, p-values are shown). A minimum of 100 cells were counted per replicate. (D) Clonogenical survival assay with continuous treatment of 16 nM olaparib and 1 μg/mL dox of the same cell lines described in (A). Data is normalized to cells treated with dox, but not treated with PARPi (n=3, mean + SEM, paired t-test, p-values are shown). (E) Same cells used in (A) and (B) were irradiated as in (B) and IF microscopy was performed to quantify BRCA1 foci (n=3, mean + SEM). A minimum of 100 cells were counted per replicate. (F) Volcano plot depicting statistical differences in binding proteins identified by MS proteomic analysis upon TY-1 IP of RPE1 hTERT TP53 -/- BRCA1 -/- cells virally reconstituted with dox-inducible BRCA1-FL or BRCA1-Δ11 1 hr post 5 Gy IR (n=3). (G) Volcano plot depicting statistical differences in binding proteins identified by MS proteomic analysis upon TY-1 IP of mock- or IR-treated (1 hr post 5 Gy) RPE1 hTERT TP53 -/- cells with an endogenous BRCA1-TY-1 tag.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α BRCA1 (Merck, OP92; 1:1000), Rabbit α BRCA1 (Merck, 07-434; 1:1000), Rabbit α BRIP1 (Sigma-Aldrich, B1310-200; 1:2000), Mouse α TUBULIN (Sigma-Aldrich, T6199; 1:5000), Rabbit α TOPBP1 (Abcam, ab2402; 1:1000), Rabbit α p-CHK1 (Cell signalling Technology, 2348S; 1:1000), Rabbit α CHK1 (Abcam, ab47574; 1:1000), Mouse α TY-1 (Diagenode, C1520054; 1:1000), and Rabbit α GAPDH (Sigma-Aldrich, G9545; 1:5000), Rabbit α p-ATR (Abcam, ab223258, 1:1000), Rabbit α ATR (Bethyl laboratories, A300-138A, 1:5000), Rabbit α p-RPA-S33 (Bethyl laboratories, A300-246A, 1:1000), Mouse α RPA (Abcam, ab2175, 1:1000), Mouse α γH2AX (Millipore, 05-636, 1:1000).

Techniques: Plasmid Preparation, Control, Western Blot, Variant Assay, Expressing, Irradiation, Microscopy, Clonogenic Cell Survival Assay, Binding Assay

Journal: bioRxiv

Article Title: DNA end-resection is stimulated by an interaction between BRCA1 exon 11 and TOPBP1

doi: 10.64898/2026.01.09.698382

Figure Lengend Snippet: (A) Cell lysates from RPE1 hTERT TP53 -/- BRCA1 -/- cells virally reconstituted with dox-inducible BRCA1-FL, BRCA1-Δ11 or EV control were subjected to TY-1 IP 1 hr post 5 Gy IR or noIR and analyzed by western blotting. (B) Volcano plot depicting statistical differences in binding proteins identified by MS proteomic analysis upon TY-1 IP of RPE1 hTERT TP53 -/- BRCA1 -/- cells virally reconstituted with a dox-inducible BRCA1-FL or BRCA1-R1699Q 1 hr post 5 Gy IR (n=3). (C) Graph showing TOPBP1 LFQ intensity from the proteomic analysis of TY-1 IPs 1 hr upon 5 Gy IR of RPE1 hTERT cell lines TP53 -/- BRCA1 -/- virally reconstituted with BRCA1-FL, BRCA1-R1699Q and BRCA1-Δ11 (n=3, mean + SEM). (D) Schematic diagram of BRCA1-FL, BRCA1-Δ11, BRCA1 harboring a R1699Q mutation (RQ) or a double Δ11 and R1699Q mutation (Δ11, RQ) (left). Lysates of the indicated cell lines were harvested and IP’ed using a TY-1 antibody and analyzed by western blotting 1 hr post 5 Gy.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α BRCA1 (Merck, OP92; 1:1000), Rabbit α BRCA1 (Merck, 07-434; 1:1000), Rabbit α BRIP1 (Sigma-Aldrich, B1310-200; 1:2000), Mouse α TUBULIN (Sigma-Aldrich, T6199; 1:5000), Rabbit α TOPBP1 (Abcam, ab2402; 1:1000), Rabbit α p-CHK1 (Cell signalling Technology, 2348S; 1:1000), Rabbit α CHK1 (Abcam, ab47574; 1:1000), Mouse α TY-1 (Diagenode, C1520054; 1:1000), and Rabbit α GAPDH (Sigma-Aldrich, G9545; 1:5000), Rabbit α p-ATR (Abcam, ab223258, 1:1000), Rabbit α ATR (Bethyl laboratories, A300-138A, 1:5000), Rabbit α p-RPA-S33 (Bethyl laboratories, A300-246A, 1:1000), Mouse α RPA (Abcam, ab2175, 1:1000), Mouse α γH2AX (Millipore, 05-636, 1:1000).

Techniques: Control, Western Blot, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: DNA end-resection is stimulated by an interaction between BRCA1 exon 11 and TOPBP1

doi: 10.64898/2026.01.09.698382

Figure Lengend Snippet: (A) Schematic diagram of BRCA1 exon 11 deletion mutants used in this panel (top). Cell lysates from RPE1 hTERT TP53 -/- BRCA1 -/- cells virally reconstituted with dox-inducible BRCA1-FL, the indicated exon 11 deletion mutants or EV control were subjected to TY-1 IP 1 hr post 5 Gy IR and analyzed by western blotting (bottom). (B) Schematic diagram of BRCA1 exon 11 C-terminal deletion mutants used in this panel (top). Lysates of the RPE1 hTERT TP53 -/- BRCA1 -/- cells virally reconstituted with the indicated dox-inducible TY-1 tagged deletion mutants cells were subjected to TY-1 IP 1 hr post 5 Gy IR and analyzed by western blotting (bottom). (C) Schematic diagram of BRCA1-SQ sites in the C-terminus of exon 11 (top). IR-irradiated lysates (1 hr post 5 Gy IR) of the indicated cells virally reconstituted with dox-inducible BRCA1-FL or BRCA1-FL in which 4 SQ sites in the C-terminus of exon 11 were mutated to alanines (SA) were immunoprecipitated using a TY-1 antibody and analyzed by western blotting (bottom). (D) Schematic diagram of TOPBP1 single or tandem BRCT deletion mutants used in this study (top). IR-irradiated lysates (1 hr post 5 Gy IR) of RPE1 hTERT cell lines TP53 -/- BRCA1 -/- virally reconstituted with dox-inducible BRCA1-FL and the indicated flag-tagged TOPBP1 deletion mutants were immunoprecipitated using a FLAG antibody and analyzed by western blotting (bottom).

Article Snippet: The following primary antibodies were used for western blotting: Mouse α BRCA1 (Merck, OP92; 1:1000), Rabbit α BRCA1 (Merck, 07-434; 1:1000), Rabbit α BRIP1 (Sigma-Aldrich, B1310-200; 1:2000), Mouse α TUBULIN (Sigma-Aldrich, T6199; 1:5000), Rabbit α TOPBP1 (Abcam, ab2402; 1:1000), Rabbit α p-CHK1 (Cell signalling Technology, 2348S; 1:1000), Rabbit α CHK1 (Abcam, ab47574; 1:1000), Mouse α TY-1 (Diagenode, C1520054; 1:1000), and Rabbit α GAPDH (Sigma-Aldrich, G9545; 1:5000), Rabbit α p-ATR (Abcam, ab223258, 1:1000), Rabbit α ATR (Bethyl laboratories, A300-138A, 1:5000), Rabbit α p-RPA-S33 (Bethyl laboratories, A300-246A, 1:1000), Mouse α RPA (Abcam, ab2175, 1:1000), Mouse α γH2AX (Millipore, 05-636, 1:1000).

Techniques: Control, Western Blot, Irradiation, Immunoprecipitation

Journal: bioRxiv

Article Title: DNA end-resection is stimulated by an interaction between BRCA1 exon 11 and TOPBP1

doi: 10.64898/2026.01.09.698382

Figure Lengend Snippet: (A) RPE1 hTERT TP53 -/- BRCA1 -/- cell lines virally reconstituted with dox-inducible FL BRCA1, an EV control or BRCA1-Δ11 together with siRNA resistant FL TOPBP1 were transfected with a control (siC) or TOPBP1 -targetting siRNA (siTOPBP1), followed by IF microscopy to analyze p-RPA and BrdU foci formation 4 hr post 10 Gy IR. Left panel shows representative microscopy images of p-RPA and BrdU foci. Upper right panel shows western blotting analyses of the cells mentioned above and lower right panel shows quantification of p-RPA foci (n=4/5, mean + SEM, paired t-test, p-values are shown). A minimum of 100 cells were counted per replicate. Quantification of BrdU signal is shown in figure S5c. (B) TP53 -/- BRCA1 -/- virally reconstituted with dox-inducible TY-1 tagged FL BRCA1 wildtype or 4 x SA mutant or BRCA1-Δ11 were irradiated with 10 Gy and 4 hr post-IR analyzed by IF microscopy to detect pRPA foci formation (n=4, mean + SEM, paired t-test, p-values are shown). A minimum of 100 cells were counted per replicate. (C) Short-term proliferation assay of the cell lines described in treated with 1000 nM olaparib and dox. Data is normalized to cells treated with dox, but not olaparib (n=3, mean + SEM, paired t-test, p-values are shown). (D) Model of the TOPBP1-BRCA1 complex function during homologous recombination.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α BRCA1 (Merck, OP92; 1:1000), Rabbit α BRCA1 (Merck, 07-434; 1:1000), Rabbit α BRIP1 (Sigma-Aldrich, B1310-200; 1:2000), Mouse α TUBULIN (Sigma-Aldrich, T6199; 1:5000), Rabbit α TOPBP1 (Abcam, ab2402; 1:1000), Rabbit α p-CHK1 (Cell signalling Technology, 2348S; 1:1000), Rabbit α CHK1 (Abcam, ab47574; 1:1000), Mouse α TY-1 (Diagenode, C1520054; 1:1000), and Rabbit α GAPDH (Sigma-Aldrich, G9545; 1:5000), Rabbit α p-ATR (Abcam, ab223258, 1:1000), Rabbit α ATR (Bethyl laboratories, A300-138A, 1:5000), Rabbit α p-RPA-S33 (Bethyl laboratories, A300-246A, 1:1000), Mouse α RPA (Abcam, ab2175, 1:1000), Mouse α γH2AX (Millipore, 05-636, 1:1000).

Techniques: Control, Transfection, Microscopy, Western Blot, Mutagenesis, Irradiation, Proliferation Assay, Homologous Recombination

Journal: Cell Death and Differentiation

Article Title: Splicing controls the ubiquitin response during DNA double-strand break repair

doi: 10.1038/cdd.2016.58

Figure Lengend Snippet: Recruitment of DNA repair factors to double-strand breaks is impaired in splicing-deficient cells. ( a ) HeLa Luc-I or Luc cells were exposed to DMSO (control), 100 nM pladienolide B or 50 μ M isoginkgetin for 1–16 h. After normalization to the corresponding DMSO value, the ratio between Luc-I and Luc luciferase activity is presented as a percentage. Means±S.D. are shown, n =4. ( b ) U2OS cells were treated with pladienolide B or isoginkgetin for 2, 6 or 16 h, irradiated (6 Gy, 1 h recovery) 1 h prior to termination of the treatment, fixed and immunostained for γH2AX, MDC1, WRAP53 β , RNF168, conjugated ubiquitin recognized by the FK2 antibody, 53BP1, RAD51 or BRCA1. Nuclei were stained with DAPI in all immunofluorescence experiments. The numbers in white represent the percentage of 100–200 cells counted whose nuclei contained >10 IR-induced foci. Means±S.D. are shown, n =3. * P -value<0.05, as determined by a non-paired two-tailed Student's t -test. The ‘foci-like' accumulations RAD51 after splicing inhibition in ( b ) are not IR-induced foci, but accumulation of RAD51 in the nucleolus for unknown reasons

Article Snippet: The following antibodies were utilized for immunofluorescent staining and western blotting: mouse α - γ H2AX (cat. no. 05-636, Millipore), rabbit α - γ H2AX (cat. no. 2577, Cell Signaling, Bionordika, Stockholm, Sweden), mouse α -MDC1 (cat. no. ab50003, Abcam, Cambridge, UK), mouse α -WDR79 (clone 1F12; used for immunofluorescent staining; Abnova, cat. no. 14761-1-AP, VWR International, Stockholm, Sweden), rabbit α -WRAP53-C2 (cat. no. PA-2020-100, Innovagen AB, Lund, Sweden), mouse α -RNF8 (cat. no. sc-271462, Santa Cruz Biotechnology), rabbit α -RNF168 (cat. no. ABE367, Millipore), mouse α -ubiquitin (FK2) (cat. no. ST1200, Calbiochem, Millipore), rabbit α -53BP1 (cat. no. NB100-904, Novus Biologicals, Bio-Techne, Abingdon, UK), mouse α -BRCA1 (cat. no. sc-6954, Santa Cruz Biotechnology), rabbit α -RAD51 (cat. no. ABE257, Millipore), mouse α -Ubiquitin (cat. no. sc-8017, Santa Cruz Biotechnology), rabbit α -H2A (cat. no. ab18255, Abcam), rabbit α -H2AX (cat. no. ab11175, Abcam), rabbit α -GFP (cat. no. ab290, Abcam), mouse α -HA (cat. no. 23675, Cell Signaling), mouse α - β -actin (cat. no. A5441, Sigma-Aldrich), rabbit α -SF3B1 (cat no. ab39578, Abcam), rabbit α -RBMX (cat. no. sc-48796, Santa Cruz Biotechnology) and mouse α -PRPF8 (cat.no. ab51366, Abcam).

Techniques: Control, Luciferase, Activity Assay, Irradiation, Ubiquitin Proteomics, Staining, Immunofluorescence, Two Tailed Test, Inhibition