Cells (4 × 105) were plated, treated with PARPi and ATRi for 12 or 24 h, washed in cold PBS, and cytospun onto a glass slide at 79 × g for 5 min. Cells were treated with 0.5% Triton and fixed in cold methanol, followed by blocking in PBS with 0.1% Triton, 2% bovine serum albumin and 10% milk. Cells were incubated with primary anti-γH2AX antibody (1:500, Millipore), anti-BRCA2 (1:1000, Millipore), anti-Rad51 antibody (1:200, Santa Cruz), and anti-RPA (1:200, Thermo Fisher Scientific) overnight at 4 °C in a humidified chamber after incubation with primary anti-γH2AX antibody (1:500, Millipore) for 2 h at room temperature for double staining. Following washing with TBST (Boston Bioproduct), cells were incubated with secondary antibodies Alexa-488 anti-mouse (1:250; Jackson ImmunoResearch) and Cy3 anti-rabbit (1:250; Jackson ImmunoResearch) for 1 h at room temperature, and slides were mounted with VectaShield (DAPI included, Vector Laboratories). Staining was imaged at ×60 with a Nikon 90i microscope and quantified using the software ImageJ.
Anti rad51 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-Rad51 antibody is a laboratory tool used to detect and study the Rad51 protein. Rad51 is a key protein involved in the process of homologous recombination, which plays a crucial role in DNA repair. The antibody can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and quantify the Rad51 protein in biological samples.
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Lab products found in correlation
Alexa fluor 568 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Anti mouse alexa 488, by Jackson ImmunoResearch (1 mentions)
Primary anti γh2ax antibody, by Merck Group (1 mentions)
Anti brca2, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti survivin antibody, by R&D Systems (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Las 3000, by GE Healthcare (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti poly adp ribose polymerase parp antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti p73 antibody, by Abcam (1 mentions)
Nbp2 24737, by Novus Biologicals (1 mentions)
Anti apex1 antibody, by Novus Biologicals (1 mentions)
Antibodies against gapdh, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
10 protocols using anti rad51 antibody
1
DNA Damage Foci Quantification
2
HNSCC Cell Radiation Response Analysis
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HNSCC cells were plated in 8-well chamber slides (30,000 cells/well) in 10% growth medium containing primocin. Cells were transfected using the protocol described earlier. At 48 hours post-plating, cells were left un-radiated or irradiated with an optimal dose of radiation and were analyzed 4 h later following incubation with anti-p-H2AX antibody (1:2000 dilution) or analyzed at 12–24 h after incubation with anti-Rad51 antibody (1:50 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). This step was followed by incubation with AlexaFlour-560 or AlexaFlour-647 IgG secondary antibody (1:500 dilution, Life Technologies, Carlsbald, CA, USA). Images were captured using a 60x or 100x oil objective on a Nikon fluorescence or Olympus confocal microscope. Cells with more than five foci were counted as positive. The relative percentage of positive cells was determined in 10 fields using the formula: (Number of positive cells per field/Total number of cells)*100. Each experiment was replicated atleast 2–3 times.
3
Protein Expression Analysis in Cancer Cells
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Cancer cells were lysed in solubilization buffer (10 mmol/L Tris‐HCl, pH 7.4, 0.1% [w/v] Nonidet P‐40, 0.1% [w/v] sodium deoxycholate, 0.1% [w/v] SDS, 150 mmol/L NaCl, 1 mmol/L EDTA, and 10 μg/mL aprotinin). Lysates were subjected to SDS‐PAGE using 10% (w/v) gels under reducing conditions. The separated proteins were electrotransferred to polyvinylidene difluoride membranes using a semi‐dry blotter. Membranes were treated with 5% (w/v) skimmed milk in 10 mmol/L Tris‐HCl, pH 7.4, 0.1% (v/v) Tween20, and 150 mmol/L NaCl for 1 h. Each membrane was then reacted with an anti‐Survivin antibody (AF‐886 R&D systems, Minneapolis, MN, USA), anti‐RAD51 antibody (sc‐8349 Santa Cruz, St. Louis, MO, USA), anti‐PLK1 antibody (37‐7000 Thermo Fisher Scientific), anti‐ZNF143 antibody (H00007702‐M01 Abnova, Taipei, Taiwan), anti‐Poly (ADP‐ribose) polymerase (PARP) antibody (9542S Cell Signaling Technologies, Danvers, MA, USA), anti‐Flag antibody (F1804 Sigma‐Aldrich, St. Louis, MO, USA), and anti‐ β‐actin antibody (A5316 Sigma‐Aldrich). After washing the membranes with T‐TBS, they were reacted with a horseradish peroxidase‐conjugated secondary antibody. They were then treated with an ECL kit (GE Healthcare Biosciences) and exposed to LAS‐3000 (GE Healthcare Biosciences).
4
Rad51 Foci Formation Assay
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Immunofluorescent foci formation was assayed as we described (31 (link)). HEK-293 cells were seeded onto poly-d -lysine coverslips (neuVitro, 18-mm diameter, catalog number GG-18-PDL) and transfected with control, Exo1, EEPD1, or Exo1/EEPD1 siRNA, and 44–48 h later, cells were treated with 10 mm hydroxyurea for 0–24 h. Cells were pre-extracted with 0.5% Triton X-100/PBS for 5 min on ice and fixed with 4% paraformaldehyde for 20 min. Coverslips were blocked for 1 h with 1% BSA/PBS and incubated with primary rabbit polyclonal anti-Rad51 antibody (1:100; Santa Cruz Biotechnology (H-92) sc-8349) overnight in a wet chamber at 4 °C. Coverslips were washed four times with PBS, incubated with secondary antibody goat anti-rabbit Alexa Fluor 594 (Invitrogen) 1:500 for 1 h, washed four times with TBS, mounted, and analyzed by confocal microscopy as above.
5
Characterization of Protein Interactions
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Anti-APEX1 antibody, catalogue # NB100-101 was purchased from Novus Biologicals, Inc., (Littleton, CO). Anti-APEX2 antibody is developed by us. Anti-RAD51 antibody, catalogue # SC-8349 was purchased from Santa Cruz, Dallas, TX. Antibodies against GAPDH (catalogue # mAb2118), HP1 (catalogue # 261), and γH2A.X (Ser139, catalogue #2577) were from Cell Signaling Technology, Inc., Danvers, MA. For immunoprecipitation studies, anti-APEX1 antibody (Cat. # ab194, Abcam) and anti-p73 antibody (Cat. # NBP2-24737, Novus Biologicals) were used.
6
Radiation-Induced RAD51 Foci Formation in HeLa Cells
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HeLa cells were cultured on coverslips (Matsunami Glass, Osaka, Japan) and irradiated using an MX-80Labo (mediXtec) at a dose of 5 or 10 Gy. After 4, 6, and 24 h, the irradiated cells were harvested and fixed with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 in PBS, the cells were incubated with anti-RAD51 antibody (dilution 1:500–1000 H-92; Santa Cruz Biotechnology). Anti-cyclin A antibody (dilution 1:1000 B8; Santa Cruz Biotechnology) was used to identify the S to G2 cell cycle phases. Alexa Fluor-568-conjugated goat anti-rabbit IgG (dilution 1:1000; Thermo Fisher Scientific) and Alexa Fluor 488 goat anti-mouse IgG (dilution 1:1000, Thermo Fisher Scientific) were used as secondary antibodies. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; dilution 1:10,000, Thermo Fisher Scientific); RAD51 foci were examined under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Cells with more than five RAD51 foci were considered RAD51 focus-positive cells. The RAD51 foci were examined under a confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) with ZEISS ZEN Software (Carl Zeiss AG). The number of RAD51 foci present in the nuclei stained with DAPI and the signal of cyclin A were quantified.
7
Immunoblotting of DNA Repair Proteins
8
Isolation and Western Blot Analysis
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CD117+ and CD117-cells were isolated from β-globin/GFP mice and protein was extracted with Radio-Immunoprecipitation Assay lysis buffer. Total protein of 50–100 μg was run on SDS/polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. Antibodies used were: Anti-BRCA2 (Ab-1) mouse mAb (EMD Millipore, OP95-100 ug) at 1:10,000 anti-RAD51-antibody (Santa Cruz biotechnology, SC 8349) at 1:10,000.
9
Fluzoparib and Olaparib Cytotoxicity Assay
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Fluzoparib was provided by Jiangsu Hengrui Medicine Co. (Shanghai, China). Olaparib was purchased from Selleckchem (Houston, TX, USA). Antibodies against γH2AX, p‐CDK1, cyclin B1, caspase 8, caspase 9, cleaved caspase 3, and β‐tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti‐RAD51 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐PAR Ab and PARP universal colorimetric assay kit were purchased from Trevigen (Gaithersburg, MD, USA).
10
Quantifying DNA Repair Foci in Irradiated HeLa Cells
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HeLa cells were cultured on coverslips (Matsunami Glass, Osaka, Japan) and were irradiated using an MX-80Labo (mediXtec) at a dose of 10 Gy. After 6 and 24 h, the irradiated cells were harvested and xed with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 in phosphate-buffered saline, the cells were incubated with anti-RAD51 antibody (dilution 1:500 H-92; Santa Cruz Biotechnology), followed by the Alexa Fluor-568-conjugated goat anti-rabbit IgG (dilution 1:1000; Molecular Probes). Cell nuclei were stained with Hoechst 33342 (Molecular Probes); RAD51 foci were examined under a uorescence microscope (Leica Microsystems, Wetzlar, Germany). Cells with more than ve RAD51 foci were considered RAD51 foci-positive cells.
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