Rpa32

Manufactured by Abcam

RPA32 is a laboratory equipment product designed for research purposes. It is a component used in various experimental protocols and techniques. The core function of RPA32 is to [DETAILED DESCRIPTION NOT AVAILABLE].

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Lab products found in correlation

Rad51, by Santa Cruz Biotechnology (3 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Hydroxyurea, by Merck Group (2 mentions) Rat anti brdu, by Abcam (2 mentions) Mouse anti brdu, by BD (2 mentions) α tubulin, by Cell Signaling Technology (2 mentions) Pcdk1 y15, by Cell Signaling Technology (1 mentions) Phistone h3 s10, by Merck Group (1 mentions) Complete protease inhibitor cocktail tablet, by Roche (1 mentions) Alexa 488, by Cell Signaling Technology (1 mentions) Dna pkcs, by Abcam (1 mentions) Cleaved parp, by Merck Group (1 mentions) H2ax s139, by Merck Group (1 mentions) Dna pkcs s2056, by Abcam (1 mentions) Atm s1981, by Abcam (1 mentions) Rpa32 s4 8, by Fortis Life Sciences (1 mentions) Rpa32 s33, by Fortis Life Sciences (1 mentions) Alexa 647, by Cell Signaling Technology (1 mentions) Irdye800 conjugated, by LI COR (1 mentions) Ir680 conjugated, by LI COR (1 mentions)

13 protocols using rpa32

Cell Signaling Protein Analysis

Cited 1 time

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Whole cell lysates were prepared in cold SDS lysis buffer (10mM Tris,
2% SDS) supplemented with PhosSTOP phosphatase inhibitor and Complete
protease inhibitor cocktail tablets (Roche) as previously described (27 (link)). The following antibodies were used:
Cdk1, pCdk1 (Y15), GAPDH (Cell Signaling Technology), pHistone H3 (S10), PAR
(Millipore), Wee1 (Santa Cruz Biotechnology), pRPA32 (S4/8) (Bethyl) and RPA32
(Abcam). Immunoblots were quantitated using ImageJ (NIH).

Karnak D., Engelke C.G., Parsels L.A., Kausar T., Wei D., Robertson J.R., Marsh K.B., Davis M.A., Zhao L., Maybaum J., Lawrence T.S, & Morgan M.A. (2014). Combined inhibition of Wee1 and PARP1/2 for radiosensitization in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(19), 5085-5096.

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Comprehensive Antibody Panel for DNA Damage Response

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Primary antibodies used were from Cell Signalling unless otherwise mentioned: β-actin (Abcam #ab6276), cleaved PARP (#5625), CDK1 Y15 (#9111), CHK1 (#2360), CHK1 S345 (#2348), H2AX (#7631), H2AX S139 (Millipore #05-636), H3 (#9715), H3 S10 (#3377), RRM2 (ABNOVA #H00006241-M01), ATR (Santa Cruz #1887), ATR T1989 (Gene Tex #GTX128145), CHK2 (#2662), CHK2 T68 (Abcam #3501), KAP1 (Abcam #10484), KAP1 S824 (Abcam #133440), DNA-PKcs (Abcam #70250), DNA-PKcs S2056 (Abcam #18192), ATM (Abcam #78), ATM S1981 (Abcam #81292), RPA32 (Abcam #2175), RPA32 S4/8 (Bethyl Laboratories #A300-245A), RPA32 S33 (Bethyl Laboratories #A300-246A).
For secondary antibodies, Alexa 488 (#4408, #4412) and Alexa 647 (#4410, #4414) from Cell Signalling were used in immunostaining. IRDye800-conjugated (#925-32210, #926-33210) and IR680-conjugated (#926-68070, #926-68021) antibodies from LICOR were used in immunoblotting.

Wallez Y., Dunlop C.R., Johnson T.I., Koh S.B., Fornari C., Yates J.W., Bernaldo de Quirós Fernández S., Lau A., Richards F.M, & Jodrell D.I. (2018). The ATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo to induce pancreatic ductal adenocarcinoma regression. Molecular cancer therapeutics, 17(8), 1670-1682.

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Comprehensive DNA Damage Response Assay

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ATR, p-CHK1 (S345), RPA70, and p-histone H3 (Ser10) antibodies were obtained from Cell Signaling; ATRIP, NUP153, and RPA32 were obtained from Abcam; TPR, γH2AX (S139), and lamin A Abs were obtained from Sigma and Millipore; and Chk1 was obtained from Leica. EdU (click-IT), RPA70-, TopBP1-, and RAD17-small hairpin RNAs (shRNAs) were obtained from Invitrogen and Origene Technologies, respectively. ATR-specific inhibitor and ATR shRNA were from Dr. Oscar Capetillo (Centro Nacional de Oncologia [CNIO]) (Toledo et al., 2011 (link)); the GFP-ATR plasmid was from Dr. Randal Tibbetts (Tibbetts et al., 2000 (link)); RFP-Lamin was from Prof. Howard J. Worman (Columbia University) (Ostlund et al., 2006 (link)); and RFP-Nucleophosmin was a gift by Dr. Michelle Hill (University of Queensland). GFP-H2B and m-cherry-H2B were from IFOM. Colchicine, α-tubulin Ab, Leptomycin B, Hydroxy Urea, sorbitol, Aphidicholine, and R3306 CDK1-specific inhibitor were obtained from Sigma.

Kumar A., Mazzanti M., Mistrik M., Kosar M., Beznoussenko G.V., Mironov A.A., Garrè M., Parazzoli D., Shivashankar G.V., Scita G., Bartek J, & Foiani M. (2014). ATR Mediates a Checkpoint at the Nuclear Envelope in Response to Mechanical Stress. Cell, 158(3), 633-646.

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Western Blot Analysis of DNA Repair Proteins

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Whole cell extracts were prepared with radioimmunoprecipitation assay (RIPA) buffer. Proteins (20 μg) were separated on 10% polyacrylamide gels (Invitrogen). After transfer, membranes were probed with antibodies against RPA32, RPA70, DDB1 or DDB2 (Abcam). Antigen–antibody complexes were visualized using ECL blotting detection agent (GE Healthcare).

Guven M., Brem R., Macpherson P., Peacock M, & Karran P. (2015). Oxidative damage to RPA limits the nucleotide excision repair capacity of human cells. The Journal of investigative dermatology, 135(11), 2834-2841.

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Western Blot Analysis of DNA Repair Proteins

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Whole cell extracts and the protein concentration measurements for western blotting were performed as described previously (51 (link)). Equal amounts of protein were separated on NuPAGE Tris-Acetate or Bis-Tris gel (Invitrogen) and transferred to a PVDF membrane (0.45 μm pore size) (Invitrogen). After blocking, the membranes were incubated with appropriate primary antibodies at 4°C overnight followed by secondary antibodies at room temperature for 1 h. Imaging of protein was performed by the BIO-RAD ChemiDoc Imaging sytem.
Primary antibodies used for western blotting are listed below:
BRCA2 (Santa Cruz, sc-28235); BRCA1 (Calbiochem, OP92); RAD51 (Santa Cruz, sc-8349); CtIP (GeneTex 19E8); MRE11 (Abcam, ab33125); RPA32 (Abcam, 2175); Tubulin (Sigma, T5168). All secondary antibodies were purchased from Invitrogen.

Ahrabi S., Sarkar S., Pfister S.X., Pirovano G., Higgins G.S., Porter A.C, & Humphrey T.C. (2016). A role for human homologous recombination factors in suppressing microhomology-mediated end joining. Nucleic Acids Research, 44(12), 5743-5757.

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Immunofluorescence Staining of DNA Repair Proteins

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The following antibodies were used: 53BP1 (Abcam ab21083), RPA32 (Abcam ab2175), PCNA (Santa Cruz sc-56), mouse anti-BrdU (BD Biosciences347580), rat anti-BrdU (Abcam ab6326), DCAF14 (Novus NBP2-33883), and KU70 (Abcam ab92450).

Tirado-Class N., Hathaway C., Chung W.K, & Dungrawala H. (2022). PHIP variants associated with Chung–Jansen syndrome disrupt replication fork stability and genome integrity. Cold Spring Harbor Molecular Case Studies, 8(5), a006212.

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Immunoblotting and Immunofluorescence Assays

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Primary antibodies to cellular proteins were purchased from commercial sources: Mre11 (Novus NB100-142), Rad50 (GeneTex [13B3] GTX70228), Nbs1 (Novus NB100-143), ATM pS1981 (Epitomics 2152-1 and Abcam [EP1890Y] ab81292), ATM (Abcam [Y170] ab32420 and Epitomics 1549-1), Actin (Sigma a5441), RPA32 (Abcam ab2175 and Bethyl A300-244A), PML (Santa Cruz [PG-M3] sc-966), and FLAG (Sigma F3165 and F7425). Primary antibodies to adenoviral proteins DBP and E4orf3 were gifts from A. Levine and T. Dobner, respectively. Horseradish peroxidase-conjugated secondary antibodies for immunoblotting were purchased from Jackson Laboratories. Fluorophore-conjugated secondary antibodies for immunofluorescence were purchased from Life Technologies. The ATM kinase inhibitor KU55933 was purchased from Abcam. The proteasome inhibitor MG132 was purchased from Sigma-Aldrich.

Pancholi N.J, & Weitzman M.D. (2018). Serotype-specific restriction of wild-type Adenoviruses by the cellular Mre11-Rad50-Nbs1 complex. Virology, 518, 221-231.

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Antibody Assay for Cell Cycle Markers

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Antibodies against pChk1 (S296), Cdc6, E2F1, RPA32 and RRM2, and were purchased from Abcam; pCDK1/2 (Y15), CDT1, pChk1 (S317), Chk1, pChk2 (T68), Chk2, Cyclin A2, Cyclin E1, p DNA-PKcs (S2056), DNA-PKcs, GAPDH, Geminin, pH2AX (S139), pHH3 (S10), Ki67, MCM2, PCNA, pRb (S807/S811) and Rb from Cell Signaling Technologies; pRPA32 (S4/S8) from Bethyl Laboratories and pH2AX (S139) (clone JBW301) from Merck Millipore. Antibodies were used at the manufacturer’s recommended dilutions.

, & Massey A.J. (2016). Tumour growth environment modulates Chk1 signalling pathways and Chk1 inhibitor sensitivity. Scientific Reports, 6, 35874.

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Antibodies and Reagents for Cell Signaling

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Actin, TRADD (mouse), Lamin B, RAD51, Myc, and Sp1 antibodies were from Santa Cruz. 53BP1 antibody was from Cell signaling. Hsp90 and RPA32 antibodies were from Abcam. TRADD (human) or γH2AX (phospho-S139) antibodies were from Millipore, Cell signaling and Genetex. Phospho-JNK and JNK antibodies were from Invitrogen. The RIPK1 antibody was from BD transduction. Hydrogen Peroxide (H₂O₂), Doxorubicin (Doxo), Etoposide (Etopo), Camptothecin (Cpt), Cisplatin (CDDP), Hydroxyurea (Hu), JNK inhibitor (SP600125), NAC (N-acetyl-I-cysteine), Phleomycin (Phleo), Propidium iodide (PI) and CM-H₂DCFDA were from Sigma Aldrich.

Koo G.B., Ji J.H., Cho H., Morgan M.J, & Kim Y.S. (2017). Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair. Scientific Reports, 7, 3332.

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Immunoblotting for DNA Repair Proteins

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Cell extracts were prepared in RIPA lysis buffer (NEB), supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma). Immunoblots were performed using standard procedures. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 3-8% or 4-12% gradient gels (Invitrogen) and subsequently transferred onto PVDF membranes. The following antibodies were used in 5% milk: BRCA1 (1:1000, Santa Cruz, #sc-6954), a-Tubulin (1:1000, CellSignaling, #3873), RPA32 (1:1000, abcam, #ab2175), Vinculin (1:5000, Sigma-Aldrich, #MAB3574). Secondary antibodies (HRP-conjugated goat anti-mouse or anti-rabbit IgG, Jackson Immunochemicals) were used at a 1:5000 dilution. Immunoblots were imaged using a Curix60 (AGFA) table-top processor.

Schrempf A., Bernardo S., Arasa Verge E.A., Ramirez Otero M.A., Wilson J., Kirchhofer D., Timelthaler G., Ambros A.M., Kaya A., Wieder M., Ecker G.F., Winter G.E., Costanzo V, & Loizou J.I. (2022). POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells. Cell reports, 41(9).

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