PA1-311

antibody from Invitrogen Antibodies

Targeting: ESR2 ER-beta, Erb, NR3A2

Provider product page for PA1-311

Western blot

Immunocytochemistry

Immunohistochemistry

Gel shift

Other assay

Antibody data

Antibody Data
Antigen structure
References [106]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Other assay [28]

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Product number: PA1-311 - Provider product page
Provider: Invitrogen Antibodies
Product name: Estrogen Receptor beta Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-311 detects estrogen receptor (ER) beta from human, non-human primate, mouse, ovine and rat tissues. PA1-311 has been successfully used in Western blot, immunocytochemistry and gel shift procedures. By Western blot, this antibody detects an ~55-62 kDa protein representing ER beta from rat brain homogenate. PA1-311 efficiently supershifts rat ER beta/ER response element (vitellogenin xenopus A2 gene). The PA1-311 immunogen is a synthetic peptide corresponding to residues A(55) E P Q K S P W C E A R S L E H(70) of rat ER beta. Recombinant ER beta expressed in baculovirus system (Cat. # RP-311 and RP-312) can be purchased for control experiments in Western blot and gel shift assays. PA1-311 can be used with blocking peptide PEP-011.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 50 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

ESR2 protein structure - PA1-311 shown in red.

Supportive validation

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Supportive validation

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Experimental details: Figure 1 Fig. 1A Photomicrograph of disc tissue showing immunolocalization of ERbeta in disc cells in the annulus of a cervical disc specimen from a 49 year old female patient. Localization is cytoplasmic. Fig. 1B , negative control. (original magnification x 640).

Supportive validation

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Experimental details: Figure 4 Association of ER-mediated gene expression with receptor and coregulator cistromes. ( A ) Comparison of number of genes with at least one binding site within 20 kb and E2-upregulated or -downregulated genes. ( B ) Association of gene regulations in different cell backgrounds with binding site clusters: enrichment of binding-site clusters (C1-C11) associated with upregulated and downregulated genes in each cell background after 4 or 24 h of estradiol treatment is shown. Level of enrichment is designated by the color scale. Cluster 3.0 was used to cluster the data. Data were visualized using Java Treeview. ( C ) Clustering of genes that are regulated by E2 treatment and genes that have binding sites from different clusters: BED files for E2-regulated genes were generated using Genespring. The seqMINER enrichment based method was used to calculate the density array for the E2-regulated gene BED files and BED files obtained for each cluster from Figure 2A . Density array was clustered using Cluster 3.0 and data was visualized using Java Treeview. ( D ) The pS2 gene and associated binding sites for ERalpha, ERbeta, RIP140, and SRC3 in the three cell backgrounds. ( E ) The pS2 gene regulation by E2 (10 -13 to 10 -7 M) in the different cell backgrounds. ( F ) The pS2 gene ChIP for ERalpha, ERbeta, and coregulators in cells treated with control vehicle (0.01% ethanol) or 10 nM E2. ( G ) The PgR gene and associated binding sites in cells treated with control vehicle (0.01% et

Supportive validation

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Experimental details: 1 Estrogen receptor (ER) staining intensities of endothelial cells. Boxplot graphic of ERalpha and ERbeta. Group 1, OVX; Group 2, OVX+RL; Group 3, OVX+AV; Group 4, OVX+RL+AV; Data are presented as median. Kruskal-Wallis test was performed to determine the differences between groups.

Supportive validation

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Experimental details: Figure 1 Steroid receptor expression in purified OSCs. ( A , B ) Steroid receptor expression profile in OSCs from young adult (2-month-old) mouse ovaries by RT-PCR ( A ) and western blot analysis ( B ). Expression of beta-actin is shown as a control for equality of sample loading; +RT and -RT represent PCR of RNA samples with and without reverse transcription, respectively (the latter used to rule out target gene amplification from potential genomic DNA contamination). Adult ovarian tissue was used as a positive control, as indicated, since all three steroid receptors under investigation (ERalpha, ERbeta, PR) are widely known to be expressed in this tissue. ( C - G ) Flow cytometric analysis of ERalpha protein expression in extracellular Ddx4-positive OSCs. ( C ) ERalpha-negative control gate; ( D ) population shift for ERalpha-positive cells; ( E ) population shift for extracellular Ddx4-positive cells (see panel G for negative control gate); ( F ) extracellular Ddx4/ERalpha dual-positive cells, as shown in the upper right quadrant; ( G ) extracellular Ddx4-negative control gate. ( H ) Quantification of the percent of OSCs (extracellular Ddx4-expressing cells) dual-positive for ERalpha expression (93.8 +- 0.5%; mean +- SEM, n = 3 independent sorts).

Supportive validation

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Experimental details: Fig. 7 ERbeta receptor distribution in the TG. a ERbeta expression is found in the cytoplasm of most neurons. In the higher magnification to the right, arrow points at an ERbeta immunoreactive neuron. b Double immunohistochemistry with ERbeta and CGRP antibodies revealed in higher magnification that both ERbeta and CGRP expression is found in the same organelle, possibly the Golgi apparatus (arrow heads)

Supportive validation

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Experimental details: Fig. 9 ERalpha and ERbeta distribution in cerebral arteries. ERalpha and ERbeta immunohistochemistry of cerebral arteries revealed that both ERalpha and ERbeta expression was found in the smooth muscle cells (arrow) and the endothelial cells (arrowhead)

Supportive validation

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Experimental details: Immunofluorescence of ERbeta in pre-implantation embryos. Scattered ERbeta expression was detected in the cytoplasm as well as the nucleus (co-stained with DAPI) of 1-cell stage embryos to blastocysts. ERbeta: green; DAPI: blue. Scale bar = 50 mum (A) Unstained; (B) stained with DAPI; (C) stained with primary and secondary antibodies for ERbeta; (D) stained with only secondary antibodies for ERbeta.Representative images from three experiments using biologically different samples (n >= 15) are shown. Fig. 2

Supportive validation

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Experimental details: Figure 2 Categorization of binding sites into clusters based on ERalpha, ERbeta, SRC3, and RIP140 cistromes. ( A ) Clustering of the binding sites. seqMINER software was used for clustering based on colocalization of different factors in different cell backgrounds within a 300-bp window in both directions. ( B ) Conservation of binding sites among vertebrates: cistrome conservation tool was used to compare conservation of binding sites from different clusters among vertebrates. ( C ) Genomic location of binding sites: web-based CEAS tool was used for identifying genomic location of binding sites from each cluster.

Supportive validation

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Experimental details: Figure 6 ERalpha supports strong E2-mediated cell proliferation; ERbeta reduces the proliferative stimulation of ERalpha in ERalpha/ERbeta cells, and ERbeta by itself supports very little if any hormone-enhanced proliferation. ( A ) Cell proliferation: ERalpha, ERalpha/ERbeta, and ERbeta cells in six-well plates were treated with Veh (0.01% EtOH) or 10 nM E2 (day 0). Treatment was repeated at day 2. Cell number was monitored by MTS assay. Treatment groups were monitored in triplicate in two separate experiments. ( B ) FACS analysis: cell cycle stages were analyzed by flow cytometry using BD-FACS Canto. Cells were fixed in 70% ethanol, stained for 30 min with 20 mug/ml PI in Triton-X in the presence of DNAse-free RNAse A, and PI staining was measured. ( C ) Gene expression of four M-phase-related genes in cells treated with control vehicle or 10 nM E2 for 24 h. mRNA level in vehicle-treated ERalpha cells was set at 1.

Supportive validation

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Experimental details: 2 Distribution of estrogen receptor (ER)alpha and ERbeta immunoreactivity in the adult male rat hippocampus. (a,e) ERalpha immunoreactivity in the control rat hippocampus by anti-ERalpha antibody (MC-20): (a) CA3 region. ERalpha immunoreactivity was observed predominantly in the nucleus and to a lesser extent in the cytoplasm; (e) CA1 region. ERalpha was expressed predominantly in the nucleus and to a lesser extent in the cytoplasm. (b,f) ERalpha immunoreactivity in the control rat hippocampus by anti-ERalpha antibody (PA1-309): (b) Image of the CA3 region showing ERalpha-immunoreactivity predominantly in the nucleus and to a lesser extent in the cytoplasm; (f) Image of the CA1 region showing ERalpha immunoreactivity predominantly in the nucleus and to a lesser extent in the cytoplasm; (c,g) CA3 (c) and CA1 (g) of the control rats stained immunohistochemically with anti-ERbeta antibody (PA1-310B): (c) Image of the CA3 region of a showing ERbeta immunoreactivity mainly in the cytoplasm; (g) Image of the CA1 region. ERbeta was expressed in the cytoplasm. (d,h) CA3 (d) and CA1 (h) of control rats stained immunohistochemically with anti-ERbeta antibody (PA1-311): (d) Image of the CA3 region showing ERbeta immunoreactivity mainly in the cytoplasm; (h) Image of the CA1 region. ERbeta was expressed in the cytoplasm. (i-l) Images of the CA1 region with neuronal loss in the rats on day 21 after status epilepticus stained immunohistochemically with anti-ERalpha antibody (MC-20),

Supportive validation

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Experimental details: Figure 3 RNF146 expression induced by rhododendrin is mediated by ERbeta activation. ( A ) Quantification of relative RNF146 mRNA expression levels (normalized to those of GAPDH) in SH-SY5Y cells treated with rhododendrin (10 µM, 37 h) in the presence or absence of the ER inhibitor tamoxifen (1 µM, 8 h) determined by RT-qPCR ( n = 3). ( B ) Representative Western blot showing that rhododendrin (10 µM, 37 h)-mediated induction of RNF146 expression is blocked by tamoxifen treatment (1 µM, 8 h). ( C ) Quantification of relative RNF146 expression levels (normalized to those of beta-actin) in the experimental groups in panel B ( n = 3). ( D ) Quantification of relative RNF146 expression levels (normalized to those of GAPDH) in SH-SY5Y cells treated with rhododendrin (10 µM, 37 h) determined by RT-qPCR. ERbeta expression was knocked down by CRISPR-cas9-mediated deletion of ERbeta (85 h). sgRNA to EGFP was used as CRISPR-cas9 transfection control ( n = 3). ( E ) Representative Western blot showing that rhododendrin (10 µM, 37 h)-mediated induction of RNF146 expression is abolished by CRISPR-cas9 mediated deletion of ERbeta (85 h). ( F ) Quantification of relative RNF146 and ERbeta expression levels (normalized to those of beta-actin) in the experimental groups in panel E ( n = 3). ( G ) Representative immunofluorescence images showing nuclear translocation of ERbeta in response to rhododendrin treatment (37 h) in SH-SY5Y cells. ( H ) Relative dist

Supportive validation

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Experimental details: Figure 4 Rhododendrin prevents 6-OHDA cell toxicity via ERbeta activation. ( A ) Assessment of cell viability in SH-SY5Y cells transfected with CRISPR-cas9 construct targeting ERbeta (85 h) and subsequently treated with rhododendrin (10 µM, 37 h) or DMSO as control determined by a trypan blue exclusion assay. Oxidative stress induced cell death was induced by treatment with 6-OHDA (70 uM, 16 h) ( n = 6). ( B ) Representative Western blot of PAR, RNF146, and ERbeta in SH-SY5Y cells with the indicated transfections and treatments. SH-SY5Y cells were transfected with CRISPR-cas9 constructs (85 h) followed by 37 h treatment of DMSO or 10 uM rhododendrin for RNF146 induction. 6-OHDA (70 uM) challenge for PAR activation was given for 30 min. Quantification of relative ERbeta ( C ), RNF146 ( D ), and PAR-conjugated proteins ( E ) expression levels in the experimental groups in panel B. Values are normalized to those of beta-actin ( n = 3). Quantified data are expressed as mean +- SEM. Statistical significance was determined by ANOVA test with Tukey's post-hoc analysis, ** p < 0.01, and *** p < 0.001.