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Ab175933

Manufactured by Abcam
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Ab175933 is a laboratory equipment product manufactured by Abcam. It is a core lab tool designed for a specific function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Ab2175, by Abcam (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) A11008, by Thermo Fisher Scientific (1 mentions) A21127, by Thermo Fisher Scientific (1 mentions) A11001, by Thermo Fisher Scientific (1 mentions) Tirf microscope, by Zeiss (1 mentions) Goat anti mouse igg h l alexafluor 546, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions) Bz x710 microscope, by Keyence (1 mentions) Ab23750, by Abcam (1 mentions) Ab19304, by Abcam (1 mentions) Ab14683, by Abcam (1 mentions) Sc 365684, by Santa Cruz Biotechnology (1 mentions) Eclipse 80i microscope, by Nikon (1 mentions) Ab5694, by Abcam (1 mentions) Polyperoxidase anti rabbit igg, by ZSGB-BIO (1 mentions) Ab87277, by Abcam (1 mentions) Ab191217, by Abcam (1 mentions) Ab6326, by Abcam (1 mentions)

14 protocols using ab175933

1

Immunofluorescence Microscopy of DNA Damage Markers

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Cells were cultured on chamber slides and fixed with 4% paraformaldehyde when harvested. Cells were permeabilized and blocked as described earlier for TIFs. The primary antibodies used included gamma-H2AX (Bethyl, A300-081), pRPA32 (Bethyl, A300-245) and 53BP1 (Abcam, ab175933). Secondary antibodies conjugated to an appropriate fluorophore were used to detect the proteins of interest (Invitrogen, A21127, A11001, A11008 and A31572). Cells were counterstained with DAPI to determine the nuclear localization of the protein of interest. Images were acquired with a Zeiss TIRF microscope in a blinded fashion and analyzed with FIJI software.
Stroik S., Kurtz K., Lin K., Karachenets S., Myers C.L., Bielinsky A.K, & Hendrickson E.A. (2020). EXO1 resection at G-quadruplex structures facilitates resolution and replication. Nucleic Acids Research, 48(9), 4960-4975.
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2

Quantifying 53BP1 Foci in Cyclin A-Negative Cells

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For 53BP1/Cyclin A staining, cells on coverslips were fixed for 15 min in 4% paraformaldehyde followed by 5 min in ice-cold methanol. Fixed cells were then permeabilized in 0.5% Triton X-100, blocked for 1 h in 2% BSA, 0.2% Triton X-100 at RT, and incubated for 2 h at RT with primary antibodies (rabbit anti-53BP1 (Abcam ab175933, 1:200 dilution) and mouse anti-Cyclin A2 (Calbiochem CC17, 1:100 dilution)). Coverslips were washed in PBS, incubated for 45 min in secondary antibodies (Goat anti-mouse IgG (H+L) Alexa Fluor 546 (Thermo Fisher A11003, 1:350 dilution) and Goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Thermo Fisher A11008, 1:350 dilution)) washed again with PBS, and mounted onto slides using Vectashield with DAPI. Imaging was performed using Keyence BZ-X710 microscope. A minimum of 300 Cyclin A-negative cells were analyzed for 53BP1 foci from three independent experiments.
Coleman K.E., Yin Y., Lui S.K., Keegan S., Fenyo D., Smith D.J., Rothenberg E, & Huang T.T. (2022). USP1-trapping lesions as a source of DNA replication stress and genomic instability. Nature Communications, 13, 1740.
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3

Immunohistochemical and Immunofluorescence Analysis of Tumor Markers

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Tumor tissues were fixed with 4% paraformaldehyde and then sectioned into 4 μm of sections. IHC was performed according to protocols of the manufacturor. The sections were incubated with rabbit anti-MMP2, MMP9, p-ATM, GLUT1, PKM2 and TGFβ1 polyclonal antibody (1:200, Bioworld) overnight at 4 °C. Then, the sections were sequentially incubated with polyperoxidase-anti-rabbit IgG (ZSBiO) for 30 min at 37 °C, then stained with diaminobenizidine.
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
Sun K., Tang S., Hou Y., Xi L., Chen Y., Yin J., Peng M., Zhao M., Cui X, & Liu M. (2019). Oxidized ATM-mediated glycolysis enhancement in breast cancer-associated fibroblasts contributes to tumor invasion through lactate as metabolic coupling. EBioMedicine, 41, 370-383.
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4

Antibody-based Protein Detection in DNA Damage Response

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The antibodies anti-RECQ1 (ab151501, 1:200 dilution), anti-53BP1 (ab175933, 1:200 dilution), anti-RPA (ab2175, 1: 1000 dilution), anti-PARP1 (ab191217, 1:1000 dilution) and anti-BrdU (ab6326, 1:200), anti-RPA2-pS4/S8 (ab87277, 1:200 dilution) were purchased from Abcam. Antibodies anti-γH2AX (2577, 1:800 dilution), α-Tubulin (2144, 1:1000 dilution) and PCNA antibody (2586, 1:200 dilution) were purchased from Cell Signaling. Mouse anti-BrdU (347580, 1:40) was purchased from BD Biosciences. AF647 (A-21247, 1:1000) and AF488 (A-11001, 1:1000) were purchased from Thermo Fisher Scientific. BMN673 (S7048, Talazoparib) was purchased from Selleck Chemicals.
Zhang J., Lian H., Chen K., Pang Y., Chen M., Huang B., Zhu L., Xu S., Liu M, & Zhong C. (2021). RECQ1 Promotes Stress Resistance and DNA Replication Progression Through PARP1 Signaling Pathway in Glioblastoma. Frontiers in Cell and Developmental Biology, 9, 714868.
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5

Palbociclib Signaling Pathway Analysis

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Palbociclib (Selleck Chemicals, Shanghai, China); Antibodies specific for pRB (Ser780, #9307, 1:3000), RB (#9309, 1:3000), CDK4 (#12790, 1:3000), CDK6 (#3136, 1:3000) and p16 (#80772, 1:3000) were obtained from Cell Signaling Technologies. Antibodies against γ-histone-H2AX (ab124781, 1:300), 53BP1 (ab175933, 1:300) and β-actin (ab8227, 1:3000) were ordered from Abcam (Abcam, Cambridge, UK). ECL luminescence reagent (abs920) was obtained from Absin (Absin Bioscience Inc., Shanghai, China).
Xie X., Zheng W., Chen T., Lin W., Liao Z., Liu J, & Ding Y. (2019). CDK4/6 Inhibitor Palbociclib Amplifies the Radiosensitivity to Nasopharyngeal Carcinoma Cells via Mediating Apoptosis and Suppressing DNA Damage Repair. OncoTargets and therapy, 12, 11107-11117.
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6

Immunofluorescence Analysis of Pancreatic Markers

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Antigen retrieval was performed on deparaffinized sections, and primary antibodies were used as follows: MIST1 (D208R, Cell Signaling, 1:200 dilution), aquaporin5 (AQP5) (sc9890, Santa Cruz, 1:200), sodium/potassium/chloride transporter (NKCC1) (D208R, Cell Signaling, 1:100), cytokeratin 7 (CK7) (ab9021, Abcam, 1:250), cytokeratin 5 (CK5) (905501, Biolegend, 1:200), Ki67 (550609, BD Biosciences, 1:200), E-cadherin (610181, BD Biosciences or ab40772, Abcam, 1:400), 53BP1 (ab175933, Abcam, 1:200), and vimentin (MA5-11883, Invitrogen, 1:200). Secondary antibodies used included Alexa Fluor donkey antimouse immunoglobulin G (IgG) 488 and 594; donkey antirabbit IgG 488, 594, and 647; and donkey antigoat IgG 594. Nuclei were stained with DAPI, 1:500 (Thermo Fisher Scientific). Fluorescent images were acquired using a Leica TCS SP5 confocal system and processed with ImageJ.
Luitje M.E., Israel A.K., Cummings M.A., Giampoli E.J., Allen P.D., Newlands S.D, & Ovitt C.E. (2020). Long-Term Maintenance of Acinar Cells in Human Submandibular Glands After Radiation Therapy. International journal of radiation oncology, biology, physics, 109(4), 1028-1039.
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7

Analyzing DNA Damage Response Markers

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M059J and M059K cells were cultured in six-well plates and transfected with anti-miR-1193 or anti-miR-NC. After 3 days of incubation, cells were washed with PBS and fixed with 4% formaldehyde. Cells were permeabilized with Triton X-100 (0.05%) for 10 min, blocked with 3% BSA in PBS and then incubated overnight at 4 °C with primary antibodies (anti-53BP1, Abcam, ab175933, 1:200 dilution; anti-RPA, Abcam, ab2175, 1: 200 dilution; and anti-γH2AX, Cell Signaling, #2577, 1:800 dilution). Next, cells were washed and incubated with the corresponding AF488- or AF647-conjugated secondary antibody. Finally, cells were washed with PBST and stained with DAPI for 10 min at RT. Images of the mounted slides were acquired with a Zeiss Axiovert 200 M microscope.
Zhang J., Jing L., Tan S., Zeng E.M., Lin Y., He L., Hu Z., Liu J, & Guo Z. (2020). Inhibition of miR-1193 leads to synthetic lethality in glioblastoma multiforme cells deficient of DNA-PKcs. Cell Death & Disease, 11(7), 602.
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8

Proximity Ligation Assay for DNA Damage

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PLA assay was performed using a Duolink PLA kit following the manufacturer's protocol. The following antibodies were used: TRF1 (1449) and γH2AX (Millipore 05636), FLAG (Sigma M2), and 53BP1 (Abcam ab175933).
Yang Z., Takai K.K., Lovejoy C.A, & de Lange T. (2020). Break-induced replication promotes fragile telomere formation. Genes & Development, 34(19-20), 1392-1405.
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9

Immunofluorescence Analysis of DNA Damage Markers

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polκ (ab57070, Abcam, 1:500), p-S6 (5364, Cell Signaling, 1:1000), γH2AX (2577, Cell Signaling, 1:200), 53BP1 (ab175933, Abcam, 1:200), p-Chk1 (2348, Cell Signaling, 1:100), p-Chk2 (2661, Cell signaling, 1:500), anti-mouse Alexa-488 (4408, Cell signaling, 1:1000), anti-rabbit Alexa-488 (4412, Cell Signaling, 1:1000), anti-rabbit Alexa-594 (8889, Cell Signaling, 1:1000)
Temprine K., Campbell N.R., Huang R., Langdon E.M., Simon-Vermot T., Mehta K., Clapp A., Chipman M, & White R.M. (2020). Regulation of the error-prone DNA polymerase Polκ by oncogenic signaling and its contribution to drug resistance. Science signaling, 13(629), eaau1453.
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10

Immunofluorescent Detection of 53BP1

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BGC-823 cells were fixed with paraformaldehyde (E672002-0500, Sangon), washed, and permeabilized in 0.5% Triton X-100 (T8787-100ML, Sigma). After blocking, the cells were immunostained with 53BP1 antibody (1 : 1000; ab175933, Abcam) and then immunostained with Alexa Fluor 555-labeled anti-rabbit IgG fluorescent antibody (1 : 2000, ab150078, Abcam). Prepared PBST solution was added for washing, followed by dehydration with ethanol. Finally, 4′,6-diamidino-2-phenylindole (DAPI; H-1200-10, Vectorlabs) was added and sealed, and the results were observed under a fluorescence microscope.
Yang Q., Nie Z., Zhu Y., Hao M., Liu S., Ding X., Wang F., Wang F, & Geng X. (2023). Inhibition of TRF2 Leads to Ferroptosis, Autophagic Death, and Apoptosis by Causing Telomere Dysfunction. Oxidative Medicine and Cellular Longevity, 2023, 6897268.
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