Erα hc 20

Manufactured by Santa Cruz Biotechnology

Sourced in United States

ERα (HC-20) is a nuclear receptor that functions as a transcription factor. It is the primary receptor for the steroid hormone estrogen and plays a crucial role in regulating gene expression related to various physiological processes.

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Lab products found in correlation

Lightcycler 480, by Roche (1 mentions) Upl assays, by Roche (1 mentions) Goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions) Ar n 20, by Santa Cruz Biotechnology (1 mentions) Pr h190, by Santa Cruz Biotechnology (1 mentions) 17β estradiol e2, by Merck Group (1 mentions) α tubulin dm1a, by Merck Group (1 mentions) Flag tag m2, by Merck Group (1 mentions) Tunicamycin, by Merck Group (1 mentions) 4 hydroxytamoxifen, by Merck Group (1 mentions) Ab1791, by Abcam (1 mentions) Gapdh ab9484, by Abcam (1 mentions) Vimentin v9, by Abcam (1 mentions) Erk2 c 14, by Santa Cruz Biotechnology (1 mentions) Perk1 2 e 4, by Santa Cruz Biotechnology (1 mentions) E cadherin h 108, by Santa Cruz Biotechnology (1 mentions) Peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions) Axio imager z1 apotome microscope, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Hif 2α, by Abcam (1 mentions)

Close spelling variants of this product

HC-20 (21 citations) Anti- ERα HC-20 (5 citations) Antihuman ERα (HC-20) antibody (sc-543) (2 citations) ERα (HC-20) and rabbit IgG (sc2027) antibodies (2 citations) Rabbit HC-20 ERα antibody (2 citations) ERα antibody HC-20 (2 citations) ERα HC-20 antibody: sc-543 (2 citations)

11 protocols using erα hc 20

ChIP-qPCR Analysis of ER-Ligand Interactions

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ChIP was performed as described [66 (link)] from MCF-7 cells treated with ERα ligands for various times with the following antibodies: ERα HC-20 (Santa Cruz Biotechnology sc-543 (Dallas, TX, USA)), rabbit IgG (Cedarlane 011-000-003 (Burlington, ON, Canada)), SUMO2/3 (Cedarlane M114-3), or mouse IgG (Cedarlane 015-000-003). The abundance of immunoprecipitated DNA fragments was quantified by qPCR (Light Cycler 480, Roche) with UPL assays (Roche) (Suppl. Table 5). Results were analyzed by the Percent Input Method.

Traboulsi T., El Ezzy M., Dumeaux V., Audemard E, & Mader S. (2018). Role of SUMOylation in differential ERα transcriptional repression by tamoxifen and fulvestrant in breast cancer cells. Oncogene, 38(7), 1019-1037.

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Immunohistochemical Detection of Estrogen and Progesterone Receptors

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ERα (HC-20) and PR(AB-52) were purchased from Santa Cruz Biotechnology (catalogs #SC-543 and #SC-810, respectively, Santa Cruz, CA). Both antibodies were diluted 1:400 in 2.5% horse serum (in phosphate buffer saline) and hybridized to tissue sections overnight at 4 °C. Frozen tissue sections were fixed in 4% paraformaldehyde for 10 min followed by three 3-min washes in phosphate buffer saline, one 5 min incubation in 3% H₂O₂, and a 1-h incubation in 2.5% horse serum before incubation with the primary antibodies. The ImmPRESS Excel Staining kit, anti-rabbit Ig (catalog #MP-7601, Vector Laboratories, Burlingame, CA) was used for ERα according to manufacturer's instructions. Goat anti-mouse secondary antibody (catalogs # A11001, Invitrogen, Grand Island, NY. 1:400 in 2.5% horse serum, 1 h) was used as secondary antibody for PR immunostaining.

Liu Y., Yen H.Y., Austria T., Pettersson J., Peti-Peterdi J., Maxson R., Widschwendter M, & Dubeau L. (2015). A Mouse Model That Reproduces the Developmental Pathways and Site Specificity of the Cancers Associated With the Human BRCA1 Mutation Carrier State. EBioMedicine, 2(10), 1318-1330.

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Comprehensive Protein Expression Analysis

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ZR-75-1, T-47D, MCF-7, MDA-MB-231, BT-20 and MDA-MB-453 cells were seeded in 6 well plates at 5 × 10⁵ cells/well in phenol red free RPMI 1640 containing 5 to 10 % hormone stripped FBS (Sigma-Aldrich), in the proportions indicated for each cell type above. Hormone stripped treatment medium was supplemented with 10nM estrogen where indicated. After 72 h, medium was replaced with the indicated hormone treatment for the specified time. Cells were lysed, protein concentration assessed, electrophoresed and transferred to Hybond-C membrane as previously described [31 (link)]. Membranes were probed using AR-N20, PR-H190, ERα-HC20, CTSD-H75, FKBP5-H100 (Santa Cruz Biotechnology, CA), calnexin (CANX, Thermo Scientific, VIC, Australia), and anti-tubulin alpha (TUBA, Millipore, VIC, Australia) and detected as previously described [31 (link)].

Need E.F., Selth L.A., Trotta A.P., Leach D.A., Giorgio L., O’Loughlin M.A., Smith E., Gill P.G., Ingman W.V., Graham J.D, & Buchanan G. (2015). The unique transcriptional response produced by concurrent estrogen and progesterone treatment in breast cancer cells results in upregulation of growth factor pathways and switching from a Luminal A to a Basal-like subtype. BMC Cancer, 15, 791.

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Estrogen Receptor Signaling Assay

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17β-estradiol (E2), 4-hydroxytamoxifen (OHT), and tunicamycin were purchased from Sigma-Aldrich. E2 and OHT were dissolved in ethanol, tunicamycin in dimethyl sulfoxide (DMSO). Primary antibodies were the following: against the FLAG tag (M2, Sigma Aldrich), α-tubulin (DM1A, Sigma Aldrich), histone H3 (ab1791, Abcam), pERK1/2 (E-4, Santa Cruz Biotech), ERK2 (C-14, Santa Cruz Biotech), GAPDH (ab9484, Abcam), ERα (HC-20, Santa Cruz Biotech), GPER (LS-A4272, LifeSpan Biosciences), vimentin (V9, Abcam), and E-cadherin (H-108, Santa Cruz Biotech).

Pupo M., Bodmer A., Berto M., Maggiolini M., Dietrich P.Y, & Picard D. (2017). A genetic polymorphism repurposes the G-protein coupled and membrane-associated estrogen receptor GPER to a transcription factor-like molecule promoting paracrine signaling between stroma and breast carcinoma cells. Oncotarget, 8(29), 46728-46744.

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ChIP Assay with Hormone Treatments

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Cells were hormone-deprived as described above prior to treatment with 0.1 % EtOH, 1 nM E2, 1 μM ICI, 100 nM P4, or 1 μM RU486 for 45 minutes. ChIP experiments were performed as described previously [20 (link)] with minor modifications:

Nuclei were extracted prior to sonication by resuspending the fixed cell pellet in nuclei preparation buffer (5 mM 1,4-piperazinediethanesulfonic acid (PIPES), 85 mM KCl [pH 8.0] + 0.5 % Nonidet P-40 + protease inhibitor) with rotation at 4 °C for 30 minutes. Nuclei were then pelleted and lysed and/or sonicated as described.

SDS was omitted from buffers Tris-sucrose-ethylenediaminetetraacetic acid I (TSEI) and TSEII.

Carrier molecules were added during immunoprecipitation [21 (link)].

In immunoprecipitation experiments, we used ERα (HC-20) and rabbit immunoglobulin G (sc2027) antibodies (Santa Cruz Biotechnologies, Dallas, TX, USA). PCR was performed as described above, normalized to percentage input. Primer sequences are available in Additional file 2.

Sikora M.J., Jacobsen B.M., Levine K., Chen J., Davidson N.E., Lee A.V., Alexander C.M, & Oesterreich S. (2016). WNT4 mediates estrogen receptor signaling and endocrine resistance in invasive lobular carcinoma cell lines. Breast Cancer Research : BCR, 18, 92.

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Western Blotting and Immunofluorescence Analyses

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The primary antibodies used for Western blotting and immunofluorescence analyses were the following: ERα (HC-20, Santa Cruz, Santa Cruz, CA, USA, sc-543), ERK (Santa Cruz, sc94), HIF1α (Abcam, Cambridge, United Kingdom, ab2185) and HIF2α (Abcam, ab199), pAKT_Ser473 (Cell Signaling, 4060). For western blotting, the peroxidase-conjugated secondary antibodies directed against rabbit or mouse constant fragments were purchased from GE HealthCare, Chicago, IL, USA. For immunofluorescence, secondary antibodies coupled with 594 or 488 Alexa Fluor dyes were obtained from Abcam. The slides were mounted in mounting medium with DAPI (Sigma–Aldrich, Saint-Louis, MI, USA). Images were obtained with an ApoTome Axio Z1 Imager microscope (Zeiss, Göttingen, Germany) and processed with Axio Vision Software (4;8;2 SP1, Zeiss, Göttingen, Germany). Fluorescence was quantified with ImageJ software from images obtained with identical exposure time.

Jehanno C., Le Goff P., Habauzit D., Le Page Y., Lecomte S., Lecluze E., Percevault F., Avner S., Métivier R., Michel D, & Flouriot G. (2022). Hypoxia and ERα Transcriptional Crosstalk Is Associated with Endocrine Resistance in Breast Cancer. Cancers, 14(19), 4934.

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ChIP Assay in MCF-7 Cells

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ChIP assays were performed as previously described (10 (link)). Experiments were conducted in hormone-depleted MCF-7 cells treated with 0.1% Control EtOH Vehicle or 10nM E2 for 45min. Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature. Cells were washed with PBS and collected using lysis buffer supplemented with 1× complete protease inhibitor mix (Roche). Chromatin was sonicated in lysis buffer to 300–500 bp size fragments. Antibodies were incubated with cell lysates overnight for chromatin collection and then with Dynabead Protein A and G (Life Technologies) for 6 h. ChIP DNA was isolated using PCR puri cation kit (QIAGEN) per the manufacturer’s instructions and used for quantitative real-time PCR. Amounts of ChIP DNA were normalized to inputs. Antibodies used for ChIP experiments were H3K4me3 (ab8895, Abcam); H3K9me3 (ab8898, Abcam); p53 (DO-1, sc-29435); RNA Polymerase II (29634A, CTD4H8 clone, Upstate); ERα (HC-20, Santa Cruz); ERβ antibodies were a combination of CWK-F12 (produced in our lab) (25 (link)), GTX70182 (GeneTex), GR40 (Calbiochem), and PA1-311 (Af nity Bioreagents); N-CoR (sc-1609, Santa Cruz); and SMRT (sc-1610, Santa Cruz).

Lu W, & Katzenellenbogen B.S. (2017). Estrogen Receptor-β Modulation of the ERα-p53 Loop Regulating Gene Expression, Proliferation, and Apoptosis in Breast Cancer. Hormones & Cancer, 8(4), 230-242.

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Quantifying ERα Chromatin Binding

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ChIP experiments were performed as previously described [22 (link)]. Briefly, hormone-deprived WT and mutant cells were treated with veh or 1 nM E2 for 45 minutes. The immunoprecipitation was performed using ERα (HC-20) and rabbit IgG (sc2027) antibodies (Santa Cruz Biotechnologies). Quantitative (q)PCR was employed to quantify the binding enrichment using the primers shown in Additional file 1: Table S3.

Bahreini A., Li Z., Wang P., Levine K.M., Tasdemir N., Cao L., Weir H.M., Puhalla S.L., Davidson N.E., Stern A.M., Chu D., Park B.H., Lee A.V, & Oesterreich S. (2017). Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models. Breast Cancer Research : BCR, 19, 60.

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EGFR Mutation and Tamoxifen Effects

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Two lung cancer cell lines, namely, PC9 (exon 19 deletion mutation; TKI-sensitive cell line) and A549 (EGFR wild-type; TKI-resistant cell line), were maintained in Dulbecco's modified Eagle medium (DMEM) (Invitrogen) with 10% fetal calf serum and 1% penicillin/streptomycin (Invitrogen) and incubated in a humidified atmosphere of 5% CO₂ at 37°C. The antibodies used were ERα (HC-20, Santa Cruz Biotechnology), ERβ (H-150, Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology). The reagents used were Gefitinib (Gef, Astra Zeneca), Tamoxifen (TAM, Astra Zeneca), and Trypan Blue (Sigma-Aldrich).

Liu C.M., Chiu K.L., Chen T.S., Chang S.M., Yang S.Y., Chen L.H., Ni Y.L., Sher Y.P., Yu S.L, & Ma W.L. (2015). Potential Therapeutic Benefit of Combining Gefitinib and Tamoxifen for Treating Advanced Lung Adenocarcinoma. BioMed Research International, 2015, 642041.

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Culturing Breast, Prostate, and HEK293FT Cells

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Breast cancer T47D cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as the full medium condition. When stimulated with the estrogen 17β-estradiol, T47D cells were cultured in phenol red-free DMEM with 10% charcoal/dextran–treated FBS for at least 3 d after switching from the full media. Prostate cancer LNCaP cells were cultured in RPMI 1640 media supplemented with 10% FBS as the full medium condition. HEK293FT cells were grown in DMEM with 10% FBS. The antibodies were purchased from the following companies: GAPDH (FL-335; Santa Cruz; sc-25778), ERα (HC-20; Santa Cruz; sc-543), FOXA1 (Abcam; ab23738), and CTCF (EMD Millipore; 07-729).

Fei T., Li W., Peng J., Xiao T., Chen C.H., Wu A., Huang J., Zang C., Liu X.S, & Brown M. (2019). Deciphering essential cistromes using genome-wide CRISPR screens. Proceedings of the National Academy of Sciences of the United States of America, 116(50), 25186-25195.

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