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Ferdinando Auricchio - One of the best experts on this subject based on the ideXlab platform.
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Estradiol Receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody.
The EMBO Journal, 2018Co-Authors: Antimo Migliaccio, Annalisa Rotondi, Ferdinando AuricchioAbstract:Estradiol Receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified Receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified Estradiol Receptor, appears to be labeled with 32P. The Receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the Receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid Receptor in tissue. It is consistent with our previous finding that a uterus Estradiol Receptor-kinase, which confers hormone binding ability to the Estradiol Receptor, in vitro phosphorylates this Receptor exclusively on tyrosine. Calf uterus Receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the Receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, Estradiol Receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the Estradiol Receptor from calf uterus.
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tyrosine phosphorylation of Estradiol Receptor by src regulates its hormone dependent nuclear export and cell cycle progression in breast cancer cells
Oncogene, 2012Co-Authors: Gabriella Castoria, Antimo Migliaccio, Ciro Abbondanza, A De Falco, Pia Giovannelli, Maria Lombardi, Maria Vittoria Barone, C De Rosa, T Giraldi, Ferdinando AuricchioAbstract:We report that in breast cancer cells, tyrosine phosphorylation of the Estradiol Receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the Receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the Receptor in nuclei of Estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to Estradiol. Thus, independently of Estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating Receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.
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Signaling-dependent nuclear export of Estradiol Receptor controls cell cycle progression in breast cancer cells
Molecular and Cellular Endocrinology, 2009Co-Authors: Gabriella Castoria, Antimo Migliaccio, Ferdinando AuricchioAbstract:Abstract Estradiol Receptor plays a key role in breast cancer and specific hormonal therapies blocking the Receptor functions have been developed. Unfortunately, many patients become resistant to this treatment and develop metastatic breast tumors. The causes of breast tumor progression and hormonal therapy resistance are still debated. Many proteins are mislocalized in human cancers, and increasing evidence indicates that nuclear exclusion of Estradiol Receptor is involved in tumorigenesis of breast cancer cells and hormonal therapy resistance. Therefore, analysis of intracellular localization of Estradiol Receptor together with screening for specific compounds that redirect the mislocalized Receptor to the correct subcellular compartment is a very promising approach to the discovery of novel anticancer compounds. We recently dissected Estradiol Receptor nuclear export in breast cancer cells and its dependence on PI3-K. This export has a strong impact on cell cycle progression. A peptide mimicking the nuclear export sequence of Estradiol Receptor specifically traps the Receptor in nuclear compartment and blocks the S-phase entry of target cells.
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hormone dependent nuclear export of Estradiol Receptor and dna synthesis in breast cancer cells
Journal of Cell Biology, 2008Co-Authors: Maria Lombardi, Antimo Migliaccio, Gabriella Castoria, Hiroshi Yamaguchi, Ettore Appella, Maria Vittoria Barone, Rosina Di Stasio, Alessandra Ciociola, Daniela Bottero, Ferdinando AuricchioAbstract:In breast cancer cells, cytoplasmic localization of the Estradiol Receptor α (ERα) regulates Estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both Estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and Estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit Estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and Estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by Estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, Estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.
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inhibition of Estradiol Receptor src association and cell growth by an Estradiol Receptor tyrosine phosphorylated peptide
Molecular Cancer Research, 2007Co-Authors: Lilian Varricchio, Antimo Migliaccio, Gabriella Castoria, Marina Di Domenico, Hiroshi Yamaguchi, Antonietta De Falco, Pia Giovannelli, William L Farrar, Ettore Appella, Ferdinando AuricchioAbstract:This report offers direct evidence that association of the Estradiol Receptor (ER) with Src triggered by steroid agonists or growth factors controls breast and prostate cancer cell growth. This association is abolished in whole cells and in vitro by a six-amino-acid peptide that mimics the sequence around the phosphotyrosine residue in position 537 of the human ERα. The phosphorylated peptide, at nanomolar concentrations, is taken up by MCF-7 and LNCaP cells derived from human mammary and prostate cancers, respectively. In addition, to block the ER/Src interaction, the phosphopeptide inhibits Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by Estradiol or androgen or triggered by epidermal growth factor. In contrast, no inhibition of the Src-mediated epidermal growth factor action on DNA synthesis is detectable in human mammary cancer cells that do not express ER (MDA-MB231), indicating that the peptide specifically targets the ER-associated Src. Remarkably, the peptide, in contrast with classic steroid antagonists, does not interfere in ER- or androgen Receptor–dependent transcriptional activity. Nevertheless, it markedly inhibits the growth of MCF-7 cell xenografts induced in immunodepressed and Estradiol-treated mice. The present report suggests that inhibition of association of steroid Receptors with Src or other signaling effectors may have therapeutic applications for patients with ER-positive tumors. (Mol Cancer Res 2007;5(11):1213–21)
Antimo Migliaccio - One of the best experts on this subject based on the ideXlab platform.
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Estradiol Receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody.
The EMBO Journal, 2018Co-Authors: Antimo Migliaccio, Annalisa Rotondi, Ferdinando AuricchioAbstract:Estradiol Receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified Receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified Estradiol Receptor, appears to be labeled with 32P. The Receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the Receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid Receptor in tissue. It is consistent with our previous finding that a uterus Estradiol Receptor-kinase, which confers hormone binding ability to the Estradiol Receptor, in vitro phosphorylates this Receptor exclusively on tyrosine. Calf uterus Receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the Receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, Estradiol Receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the Estradiol Receptor from calf uterus.
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tyrosine phosphorylation of Estradiol Receptor by src regulates its hormone dependent nuclear export and cell cycle progression in breast cancer cells
Oncogene, 2012Co-Authors: Gabriella Castoria, Antimo Migliaccio, Ciro Abbondanza, A De Falco, Pia Giovannelli, Maria Lombardi, Maria Vittoria Barone, C De Rosa, T Giraldi, Ferdinando AuricchioAbstract:We report that in breast cancer cells, tyrosine phosphorylation of the Estradiol Receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the Receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the Receptor in nuclei of Estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to Estradiol. Thus, independently of Estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating Receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.
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Signaling-dependent nuclear export of Estradiol Receptor controls cell cycle progression in breast cancer cells
Molecular and Cellular Endocrinology, 2009Co-Authors: Gabriella Castoria, Antimo Migliaccio, Ferdinando AuricchioAbstract:Abstract Estradiol Receptor plays a key role in breast cancer and specific hormonal therapies blocking the Receptor functions have been developed. Unfortunately, many patients become resistant to this treatment and develop metastatic breast tumors. The causes of breast tumor progression and hormonal therapy resistance are still debated. Many proteins are mislocalized in human cancers, and increasing evidence indicates that nuclear exclusion of Estradiol Receptor is involved in tumorigenesis of breast cancer cells and hormonal therapy resistance. Therefore, analysis of intracellular localization of Estradiol Receptor together with screening for specific compounds that redirect the mislocalized Receptor to the correct subcellular compartment is a very promising approach to the discovery of novel anticancer compounds. We recently dissected Estradiol Receptor nuclear export in breast cancer cells and its dependence on PI3-K. This export has a strong impact on cell cycle progression. A peptide mimicking the nuclear export sequence of Estradiol Receptor specifically traps the Receptor in nuclear compartment and blocks the S-phase entry of target cells.
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hormone dependent nuclear export of Estradiol Receptor and dna synthesis in breast cancer cells
Journal of Cell Biology, 2008Co-Authors: Maria Lombardi, Antimo Migliaccio, Gabriella Castoria, Hiroshi Yamaguchi, Ettore Appella, Maria Vittoria Barone, Rosina Di Stasio, Alessandra Ciociola, Daniela Bottero, Ferdinando AuricchioAbstract:In breast cancer cells, cytoplasmic localization of the Estradiol Receptor α (ERα) regulates Estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both Estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and Estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit Estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and Estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by Estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, Estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.
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inhibition of Estradiol Receptor src association and cell growth by an Estradiol Receptor tyrosine phosphorylated peptide
Molecular Cancer Research, 2007Co-Authors: Lilian Varricchio, Antimo Migliaccio, Gabriella Castoria, Marina Di Domenico, Hiroshi Yamaguchi, Antonietta De Falco, Pia Giovannelli, William L Farrar, Ettore Appella, Ferdinando AuricchioAbstract:This report offers direct evidence that association of the Estradiol Receptor (ER) with Src triggered by steroid agonists or growth factors controls breast and prostate cancer cell growth. This association is abolished in whole cells and in vitro by a six-amino-acid peptide that mimics the sequence around the phosphotyrosine residue in position 537 of the human ERα. The phosphorylated peptide, at nanomolar concentrations, is taken up by MCF-7 and LNCaP cells derived from human mammary and prostate cancers, respectively. In addition, to block the ER/Src interaction, the phosphopeptide inhibits Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by Estradiol or androgen or triggered by epidermal growth factor. In contrast, no inhibition of the Src-mediated epidermal growth factor action on DNA synthesis is detectable in human mammary cancer cells that do not express ER (MDA-MB231), indicating that the peptide specifically targets the ER-associated Src. Remarkably, the peptide, in contrast with classic steroid antagonists, does not interfere in ER- or androgen Receptor–dependent transcriptional activity. Nevertheless, it markedly inhibits the growth of MCF-7 cell xenografts induced in immunodepressed and Estradiol-treated mice. The present report suggests that inhibition of association of steroid Receptors with Src or other signaling effectors may have therapeutic applications for patients with ER-positive tumors. (Mol Cancer Res 2007;5(11):1213–21)
Gabriella Castoria - One of the best experts on this subject based on the ideXlab platform.
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tyrosine phosphorylation of Estradiol Receptor by src regulates its hormone dependent nuclear export and cell cycle progression in breast cancer cells
Oncogene, 2012Co-Authors: Gabriella Castoria, Antimo Migliaccio, Ciro Abbondanza, A De Falco, Pia Giovannelli, Maria Lombardi, Maria Vittoria Barone, C De Rosa, T Giraldi, Ferdinando AuricchioAbstract:We report that in breast cancer cells, tyrosine phosphorylation of the Estradiol Receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the Receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the Receptor in nuclei of Estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to Estradiol. Thus, independently of Estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating Receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.
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Signaling-dependent nuclear export of Estradiol Receptor controls cell cycle progression in breast cancer cells
Molecular and Cellular Endocrinology, 2009Co-Authors: Gabriella Castoria, Antimo Migliaccio, Ferdinando AuricchioAbstract:Abstract Estradiol Receptor plays a key role in breast cancer and specific hormonal therapies blocking the Receptor functions have been developed. Unfortunately, many patients become resistant to this treatment and develop metastatic breast tumors. The causes of breast tumor progression and hormonal therapy resistance are still debated. Many proteins are mislocalized in human cancers, and increasing evidence indicates that nuclear exclusion of Estradiol Receptor is involved in tumorigenesis of breast cancer cells and hormonal therapy resistance. Therefore, analysis of intracellular localization of Estradiol Receptor together with screening for specific compounds that redirect the mislocalized Receptor to the correct subcellular compartment is a very promising approach to the discovery of novel anticancer compounds. We recently dissected Estradiol Receptor nuclear export in breast cancer cells and its dependence on PI3-K. This export has a strong impact on cell cycle progression. A peptide mimicking the nuclear export sequence of Estradiol Receptor specifically traps the Receptor in nuclear compartment and blocks the S-phase entry of target cells.
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hormone dependent nuclear export of Estradiol Receptor and dna synthesis in breast cancer cells
Journal of Cell Biology, 2008Co-Authors: Maria Lombardi, Antimo Migliaccio, Gabriella Castoria, Hiroshi Yamaguchi, Ettore Appella, Maria Vittoria Barone, Rosina Di Stasio, Alessandra Ciociola, Daniela Bottero, Ferdinando AuricchioAbstract:In breast cancer cells, cytoplasmic localization of the Estradiol Receptor α (ERα) regulates Estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both Estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and Estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit Estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and Estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by Estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, Estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.
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inhibition of Estradiol Receptor src association and cell growth by an Estradiol Receptor tyrosine phosphorylated peptide
Molecular Cancer Research, 2007Co-Authors: Lilian Varricchio, Antimo Migliaccio, Gabriella Castoria, Marina Di Domenico, Hiroshi Yamaguchi, Antonietta De Falco, Pia Giovannelli, William L Farrar, Ettore Appella, Ferdinando AuricchioAbstract:This report offers direct evidence that association of the Estradiol Receptor (ER) with Src triggered by steroid agonists or growth factors controls breast and prostate cancer cell growth. This association is abolished in whole cells and in vitro by a six-amino-acid peptide that mimics the sequence around the phosphotyrosine residue in position 537 of the human ERα. The phosphorylated peptide, at nanomolar concentrations, is taken up by MCF-7 and LNCaP cells derived from human mammary and prostate cancers, respectively. In addition, to block the ER/Src interaction, the phosphopeptide inhibits Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by Estradiol or androgen or triggered by epidermal growth factor. In contrast, no inhibition of the Src-mediated epidermal growth factor action on DNA synthesis is detectable in human mammary cancer cells that do not express ER (MDA-MB231), indicating that the peptide specifically targets the ER-associated Src. Remarkably, the peptide, in contrast with classic steroid antagonists, does not interfere in ER- or androgen Receptor–dependent transcriptional activity. Nevertheless, it markedly inhibits the growth of MCF-7 cell xenografts induced in immunodepressed and Estradiol-treated mice. The present report suggests that inhibition of association of steroid Receptors with Src or other signaling effectors may have therapeutic applications for patients with ER-positive tumors. (Mol Cancer Res 2007;5(11):1213–21)
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[54] Calmodulin-stimulated Estradiol Receptor-tyrosine kinase I
Methods in Enzymology, 2004Co-Authors: Ferdinando Auricchio, Annalisa Rotondi, Antimo Migliaccio, Gabriella Castoria, Marina Di DomenicoAbstract:Publisher Summary This chapter describes the purification and characterization of the Estradiol Receptor kinase from calf uterus cytosol, which is a member of a new class of protein kinases—namely, calmodulin-stimulated protein-tyrosine kinases. The physiological role of this enzyme is corroborated by the recent finding that the Estradiol Receptor is phosphorylated on tyrosine in whole uterus. Calf uterus Estradiol Receptor is a phosphoprotein and that phosphorylation–dephosphorylation of this Receptor is controlled by a cytosol Receptor-tyrosine kinase that activates the hormone binding, and by a nuclear Receptor-phosphotyrosine phosphatase that inactivates this binding. The Receptor is purified from calf uterus by affinity chromatography on heparin-Sepharose followed by affinity chromatography. (NH 4 ) 2 SO 4 is used to separate most of the Receptor, which is precipitated at 0-25% saturation, from the kinase. Heparin-Sepharose separates the residual Receptor and the enzyme coprecipitated by 25-50% (NH 4 ) 2 SO 4 saturation. High ionic strength and heparin drastically decrease the sedimentation coefficient of the cytosol Estradiol Receptor. Therefore, it is not possible to exclude that in intact cells, Estradiol Receptor kinase and Estradiol Receptor are loosely associated.
Hubert H. Thole - One of the best experts on this subject based on the ideXlab platform.
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Retrieval of Estradiol Receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies
Histochemistry and Cell Biology, 1996Co-Authors: Walter D. Sierralta, Hubert H. TholeAbstract:The unmasking of Estradiol Receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the Receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the Receptor's domain E, showed a preference for the cytoplasmic Receptor. In stroma, all antibodies detected more Receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more Receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the Receptor differ according to the functional uterine cell type.
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Immunogold labelling of Estradiol Receptor in MCF7 cells
Cell and Tissue Research, 1995Co-Authors: Walter D. Sierralta, Ingrid Bönig, Hubert H. TholeAbstract:The distribution of Estradiol Receptor in serial sections of Estradiol-deprived and Estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the Receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of Estradiol-deprived cells, the Receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected Receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antiReceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after Estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen Receptor in Estradiol-deprived cells and support the premise of an Estradiol-induced translocation of this ligand-dependent transcription regulator.
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Immunogold labelling of the cytoplasmic Estradiol Receptor in resting porcine endometrium
Cell and Tissue Research, 1992Co-Authors: Walter D. Sierralta, Hubert H. TholeAbstract:Serial sections of resting porcine endometrium were analyzed with the monoclonal antibody 13H2 using goat antimouse IgG/5 nm gold as secondary reagent or with either polyclonal antibodies from goat #402 or the rat monoclonal antibody H222, both in combination with protein G/12 nm gold. A modestly higher labelling of nuclei than of cytoplasm was seen only with the monoclonal antibody H222. Polyclonal #402 and monoclonal 13H2 showed fewer attachments over nuclear than over cytoplasmic areas. The highest densities of attachment and of predominantly cytoplasmic labelling were obtained with the monoclonal antibody 13H2. The results confirm the earlier assumption of a restricted accessiblity of Estradiol Receptor in the cytoplasm of resting cells for immunoreagents.
Walter D. Sierralta - One of the best experts on this subject based on the ideXlab platform.
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Retrieval of Estradiol Receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies
Histochemistry and Cell Biology, 1996Co-Authors: Walter D. Sierralta, Hubert H. TholeAbstract:The unmasking of Estradiol Receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the Receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the Receptor's domain E, showed a preference for the cytoplasmic Receptor. In stroma, all antibodies detected more Receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more Receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the Receptor differ according to the functional uterine cell type.
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Immunogold labelling of Estradiol Receptor in MCF7 cells
Cell and Tissue Research, 1995Co-Authors: Walter D. Sierralta, Ingrid Bönig, Hubert H. TholeAbstract:The distribution of Estradiol Receptor in serial sections of Estradiol-deprived and Estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the Receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of Estradiol-deprived cells, the Receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected Receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antiReceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after Estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen Receptor in Estradiol-deprived cells and support the premise of an Estradiol-induced translocation of this ligand-dependent transcription regulator.
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Immunogold labelling of the cytoplasmic Estradiol Receptor in resting porcine endometrium
Cell and Tissue Research, 1992Co-Authors: Walter D. Sierralta, Hubert H. TholeAbstract:Serial sections of resting porcine endometrium were analyzed with the monoclonal antibody 13H2 using goat antimouse IgG/5 nm gold as secondary reagent or with either polyclonal antibodies from goat #402 or the rat monoclonal antibody H222, both in combination with protein G/12 nm gold. A modestly higher labelling of nuclei than of cytoplasm was seen only with the monoclonal antibody H222. Polyclonal #402 and monoclonal 13H2 showed fewer attachments over nuclear than over cytoplasmic areas. The highest densities of attachment and of predominantly cytoplasmic labelling were obtained with the monoclonal antibody 13H2. The results confirm the earlier assumption of a restricted accessiblity of Estradiol Receptor in the cytoplasm of resting cells for immunoreagents.