Epr15656

Manufactured by Abcam

Sourced in United States

EPR15656 is a recombinant rabbit monoclonal antibody that recognizes Human TRAF2. The antibody is suitable for use in Western Blotting applications.

Automatically generated - may contain errors

Lab products found in correlation

Sp142 antibody, by Roche (3 mentions) Ar clone 441, by Leica (3 mentions) Immobilon p pvdf membrane, by Merck Group (2 mentions) Chemidoc touch imaging system, by Bio-Rad (2 mentions) Topoisomerase 1 topo1 expression clone 1d6, by Leica (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Axioplan 2, by Zeiss (1 mentions) Cellsearch cxc kit, by Menarini Silicon Biosystems (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) V9131, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Anti hsp70, by Cell Signaling Technology (1 mentions) Anti atg5, by Cell Signaling Technology (1 mentions) Anti ar v7 antibody, by Abcam (1 mentions) Anti hsp27, by Cell Signaling Technology (1 mentions) Anti psmb5, by Cell Signaling Technology (1 mentions) Anti akt antibody, by Cell Signaling Technology (1 mentions) Anti flag, by Cell Signaling Technology (1 mentions) Anti lc3b, by Cell Signaling Technology (1 mentions)

14 protocols using epr15656

Prostate Cancer Cell Line Analysis

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Cell suspensions of selected prostate cancer cell lines (22Rv1, LNCap, PC3) were spun down on glass slides (190× g, 5 min) and dried at room temperature (RT) over night. Cells were subsequently fixed and permeabilized using the respective CellSearch CXC Kit reagents (Menarini-Silicon Biosystems, Florence, Italy) and blocked with 10% AB-Serum (BioRad, Rüdigheim, Germany). Primary antibodies targeting ARV7, 4 µg/mL of clone AG10008 (unlabeled, Precision, Columbia, Maryland, USA) and 6 µg/mL EPR15656 (unlabeled, Abcam, Cambridge, United Kingdom) were tested. Secondary antibodies were applied and contained a DAPI nuclear counterstain. Secondary rabbit-anti mouse (Alexa 546, polyclonal, Thermo Fisher Scientific, Dreieich, Germany) and mouse-anti-rabbit (Alexa 546, polyclonal, Thermo Fisher Scientific, Dreieich, Germany) antibodies were used. Cytospins were covered in Prolong Gold Antifade Reagent (Thermo Fisher Scientific, Dreieich, Germany) and cover slipped for analysis. Slides were manually assessed using a fluorescence microscope (Axioplan 2, Carl Zeiss, Oberkochen, Germany).

Hille C., Gorges T.M., Riethdorf S., Mazel M., Steuber T., Von Amsberg G., König F., Peine S., Alix-Panabières C, & Pantel K. (2019). Detection of Androgen Receptor Variant 7 (ARV7) mRNA Levels in EpCAM-Enriched CTC Fractions for Monitoring Response to Androgen Targeting Therapies in Prostate Cancer. Cells, 8(9), 1067.

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Western Blot Analysis of AR-V7

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Cells were lysed with RIPA buffer (Pierce) supplemented with protease inhibitor cocktail (Roche). Protein extracts (20 μg) were separated on 7% NuPAGE Tris-Acetate gel (Invitrogen) by electrophoresis and subsequently transferred onto Immobilon-P PVDF membranes of 0.45 μm pore size (Millipore). Primary antibodies used were rabbit monoclonal anti–AR-V7 (1 in 1,000; RM7, RevMAb Biosciences), rabbit monoclonal anti–AR-V7 (1 in 1,000; EPR15656, Abcam), and mouse monoclonal anti-vinculin (1 in 200,000; V9131, Sigma-Aldrich) with species-specific secondary antibodies conjugated to horseradish peroxidase. Chemiluminescence was detected on the Chemidoc Touch imaging system (Bio-Rad).

Sharp A., Coleman I., Yuan W., Sprenger C., Dolling D., Rodrigues D.N., Russo J.W., Figueiredo I., Bertan C., Seed G., Riisnaes R., Uo T., Neeb A., Welti J., Morrissey C., Carreira S., Luo J., Nelson P.S., Balk S.P., True L.D., de Bono J.S, & Plymate S.R. (2018). Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer. The Journal of Clinical Investigation, 129(1), 192-208.

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Immunohistochemical Detection of AR-V7 Protein in CRPC

Cited 8 times

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Recent studies on in-situ detection of AR-V7 protein by IHC focused on
newly developed and validated antibodies ^{48 (link)–53 (link)}. Using
matched CSPC and CRPC tissue samples, nuclear AR-V7 protein detected by a rabbit
AR-V7 monoclonal antibody (EPR15656; Abcam, Burlingame, CA) was associated with
poor prognosis of CRPC patients after abiraterone or enzalutamide treatment
^{51 (link)}. Following detailed
characterization in clinical cohorts ^{13 (link),49 (link),54 (link)}, the EPIC CTC AR-V7 IHC test (Epic
Sciences, San Diego, CA) initially developed using the same anti-AR-V7 antibody
was recently implemented for clinical use (see companion review). In
tissue-based studies, the use of the RM7 (RevMab) antibody further established
CRPC-specific expression AR-V7 ^{48 (link),50 (link),55}. Interestingly, AR-V7 protein expression
was detected using this well-validated antibody in a subset of cells in small
cell prostate carcinoma and some salivary ductal carcinoma specimens from
untreated female patients ^{52 (link),56 (link)}. These recent studies on AR-V7
protein expression further supported that AR-V7 expression arises specifically
in a low androgen environment.

Lu C., Brown L., Antonarakis E.S., Armstrong A.J, & Luo J. (2020). Androgen Receptor Variant–Driven Prostate Cancer II: Advances in Laboratory Investigations. Prostate cancer and prostatic diseases, 23(3), 381-397.

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Protein Extraction and Immunoblotting Protocol

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Cell lysis, protein extraction, SDS-PAGE, and immunoblotting were performed as described previously (13 (link)), with a few modifications. The Luminata Western HRP Substrate chemiluminescence kit (Millipore) was used to reveal immunoblotted proteins. The rabbit polyclonal anti-Sigma1 antibody was generated in our laboratory as described elsewhere (13 (link)). The anti-β-actin and anti-HA (Y-11) antibodies were purchased from Santa Cruz Biotechnology. The rabbit anti-FLAG, anti-GAPDH, anti-LC3B, anti-ATG5, anti-p97/VCP, anti-PSMB5, anti-polyubiquitin (clone P4D1), anti-HSP90, anti-HSP70, anti-HSP27, anti-glucocorticoid receptor (GR), anti-ErbB2/HER2, anti-ErbB3/HER3, anti-Akt antibodies, and horseradish peroxidase conjugated secondary antibodies were all purchased from Cell Signaling Technology. The anti-androgen receptor (D6F11) rabbit monoclonal antibody (Cell Signaling Technology) used here was raised against an epitope in the amino-terminus of AR. The anti-ARV7 antibody (Abcam, EPR15656) was raised against an epitope specific to the ARV7 splice variant, and the anti-AR^v567es antibody (Abcam, EPR15657) was raised against an epitope specific to the AR^v567es splice variant. We confirmed the specificity of the commercial ARV7 and AR^v567es antibodies (Supplemental Figure 1).

Thomas J.D., Longen C.G., Oyer H.M., Chen N., Maher C.M., Salvino J.M., Kania B., Anderson K.N., Ostrander W.F., Knudsen K.E, & Kim F.J. (2017). Sigma1 Targeting to Suppress Aberrant Androgen Receptor Signaling in Prostate Cancer. Cancer research, 77(9), 2439-2452.

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Biomarker Quantification in Cancer Samples

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Quantification of AR staining (SP107 clone; Ventana) was performed with positivity defined as strong nuclear staining in ≥10% of tumor cells. Detection of the splice variant AR‐V7 was assessed at protein level by IHC (EPR15656; Abcam) (16 cases from Caris) and at mRNA level by anchored multiplex PCR for targeted RNA sequencing (ArcherDX) (12 cases from Thomas Jefferson University Hospital).
Positive HER2 IHC was defined as 3+ (strong membranous staining) in ≥10% of tumor cells. IHC for CD274 (PD‐L1) was performed using either 28‐8 (Agilent) or SP142 (Ventana) clones, and PD‐L1 positivity was defined as membranous expression in ≥1% cancer cells (TC).23 Additionally, PD‐L1 expression on the IC was recorded. All IHC assays were run with both positive and negative controls using fully automated staining platforms (Ventana‐Roche and DAKO‐Agilent). The assays were conducted in a CLIA/CAP/ISO15189 certified clinical laboratory (Caris Life Sciences).23

Gargano S.M., Senarathne W., Feldman R., Florento E., Stafford P., Swensen J., Vranic S, & Gatalica Z. (2019). Novel therapeutic targets in salivary duct carcinoma uncovered by comprehensive molecular profiling. Cancer Medicine, 8(17), 7322-7329.

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Comprehensive Biomarker Profiling in Extramammary Paget's Disease

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PD‐L1 expression was evaluated in the tumor (TC) and immune cells (IC) using SP142 antibody (Ventana). Any PD‐L1 expression was considered positive if either TC or IC exhibited staining. AR (clone 441, Leica Biosystems, Buffalo Grove, IL), ER (SP1 clone, Ventana, Tucson, AZ) and PR (1E2, Ventana, Tucson, AZ) were analyzed using a ≥10% threshold for nuclear positivity. HER2 (4B5 clone, Ventana) was considered positive if >10% cancer cells showed complete, circumferential (3+) expression or exhibited HER2 gene amplification (see below). Nine cases (five vulvar and four scrotal) of EMPD and four MPD were explored for the expression of the splice variant of AR (ARv7) using immunohistochemistry (EPR15656, Abcam). Three EMPD cases were tested for mismatch repair proteins: MLH1 (Clone M1, Ventana), MSH2 (Clone G219‐1129, Ventana), MSH6 (Clone 44, Cell Marque) and PMS2 (Clone EPR3947, Cell Marque). Topoisomerase 2α (Clone 3F6, Leica) expression was considered positive if cancer cells exhibited nuclear positivity in ≥10%.7

Gatalica Z., Vranic S., Krušlin B., Poorman K., Stafford P., Kacerovska D., Senarathne W., Florento E., Contreras E., Leary A., Choi A, & In G.K. (2020). Comparison of the biomarkers for targeted therapies in primary extra‐mammary and mammary Paget's disease. Cancer Medicine, 9(4), 1441-1450.

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Western Blot Analysis of AR and PSA

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Approximately 250,000 cells per well were plated in 6-well plates in RPMI supplemented with 10% CSS. After three days, the cells were collected to prepare whole cell lysates. Protein concentrations were determined by bicinchoninic acid assay and 20 μg of cell lysate per sample was run on an SDS-PAGE gradient gel (4–15%), transferred to a nitrocellulose membrane, and then probed with antibodies raised against AR (N-20, Santa Cruz Biotechnology), AR-V7 (EPR15656, Abcam), or PSA (C-19, Santa Cruz Biotechnology). Membranes were re-probed with antibodies against β-actin (AC-15, Sigma-Aldrich) as a loading control. Western blot images were captured using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, California) and then quantified using ImageJ software.

Leung J.K., Tam T., Wang J, & Sadar M.D. (2020). Isolation and Characterization of Castration-Resistant Prostate Cancer LNCaP95 Clones. Human cell, 34(1), 211-218.

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Quantification and Characterization of AR-V7+ CTCs

Cited 2 times

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CTC identification, characterization, and analysis has been described previously.^{22 (link),24 ,25 (link)} In brief, slides created from mCRPC patient samples underwent automated immunofluorescent staining for DNA, cytokeratins (CK), CD45, and AR-V7. A rabbit monoclonal anti-AR-V7 antibody (EPR15656, Abcam, Burlingame, CA) was used for all applications described herein. Fluorescent scanners and morphology algorithms were used for identification of CTCs, evaluating two slides per sample. Clinical laboratory scientists licensed in California conducted final quality control of CTC classification and subcellular biomarker localization.
AR-V7 scoring criteria were utilized as reported previously.^{22 (link),25 (link)} In short, samples with at least one CTC with an intact nucleus and nuclear-localized AR-V7 signal-to-noise ratio above a previously established and validated background intensity per two slides tested (~1 mL of blood) was scored “AR-V7-positive.” Samples without AR-V7-positive CTCs, or with no CTCs detected, were scored “AR-V7-negative.” The analytical specificity of detection in contrived CTC samples, patient CTC samples, and healthy and malignant solid tissues and blood was previously reported.^{22 (link)}

Scher H.I., Graf R.P., Schreiber N.A., Jayaram A., Winquist E., McLaughlin B., Lu D., Fleisher M., Orr S., Lowes L., Anderson A., Wang Y., Dittamore R., Allan A.L., Attard G, & Heller G. (2018). Assessment of the Validity of Nuclear-Localized Androgen Receptor Splice Variant 7 in Circulating Tumor Cells as a Predictive Biomarker for Castration-Resistant Prostate Cancer. JAMA Oncology, 4(9), 1179-1186.

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Biomarker Assessment in Tumor Samples

Cited 3 times

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PD-L1 expression was assessed in the tumor cells and immune cells using SP142 antibody (Ventana Medical Systems, Oro Valley, AZ). PD-L1 expression was considered positive if either tumor cell or immune cells exhibited any membranous or cytoplasmic staining.^{11 (link),14 (link)} AR (clone 441, Leica Biosystems, Buffalo Grove, IL) was analyzed using a 10% or higher threshold for nuclear positivity.^{12 (link)–14 (link)} The ARv7 splice variant was explored at the protein level by immunohistochemistry (EPR15656; Abcam) and at the messenger RNA level using anchored multiplex polymerase chain reaction for targeted RNA sequencing (ArcherDX).^{9 (link),11 (link)} Topoisomerase 1 (Topo1) expression (clone 1D6; Leica Biosystems, Nussloch, Germany) was scored as 0+, 1+, 2+, or 3+ depending on the staining intensity, and the percent tumor stained was also recorded. The threshold for Topo1 overexpression was a staining intensity of 2+ or higher in 30% or more of the cancer cells.^{15 (link)}

Kruslin B., Gatalica Z., Hes O., Skenderi F., Miettinen M., Contreras E., Xiu J., Ellis M., Florento E., Vranic S, & Swensen J. (2021). TERT Gene Fusions Characterize a Subset of Metastatic Leydig Cell Tumors. Clinical genitourinary cancer, 19(4), 333-338.

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Immunohistochemical Detection of AR-V7 Protein in CRPC

Cited 8 times

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