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9924
Phospho-Estrogen Receptor α Antibody Sampler Kit

Phospho-Estrogen Receptor α Antibody Sampler Kit #9924

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Western Blotting Image 1

Western blot analysis of extracts from ER-positive cell lines (MCF7, T-47D, ZR-75-1) and ER-negative cell lines (SK-BR-3 and MCF 10A) using Estrogen Receptor α (D8H8) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Chromatin IP-seq Image 2

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 minutes and 5 μl of Estrogen Receptor α (D8H8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across TFF1/pS2, a known target gene of Estrogen Receptor α (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 3

Western blot analysis of extracts from COS-1 cells expressing wild-type or mutant ER alpha, stimulated with beta-estradiol (100 nM) and EGF (100 ng/ml) for 30 minutes, using Phospho-Estrogen Receptor alpha (Ser104/106) Antibody (upper) or control ER alpha Antibody #2512 (lower). (Cell lysates provided by Dr. Simak Ali, Hammersmith Hospital, London.)

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Western Blotting Image 4

Western blot analysis of extracts from untransfected MCF-7 cells, and COS-1 cells transfected with wild-type or mutant ER alpha, stimulated with EGF and E2, using Phospho-Estrogen Receptor alpha (Ser118) (16J4) Mouse mAb (upper) or control estrogen receptor alpha antibody #2512 (lower).

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Western Blotting Image 5

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IF-IC Image 6

Confocal immunofluorescent analysis of MCF7 (left) or SK-BR-3 (right) cells using Estrogen Receptor α (D8H8) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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Chromatin IP Image 7

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 minutes and either 5 μl of Estrogen Receptor α (D8H8) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human ESR1 Promoter Primers #9673, SimpleChIP® Human pS2 Promoter Primers #9702, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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Western Blotting Image 8

Western blot analysis of extracts from serum-starved MCF7 cells, untreated, treated with β-estradiol, or treated with β-estradiol and heregulin, using Phospho-Estrogen Receptor α (Ser104/106) Antibody. A non-specific band is detected at 90 kDa.

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IHC-P (paraffin) Image 9

Immunohistochemical staining of phosphorylated estrogen receptor alpha in paraffin-embedded human breast carcinoma showing nuclear localization, using Phospho-Estrogen Receptor alpha (Ser118) (16J4) Mouse mAb.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Estrogen Receptor α (D8H8) Rabbit mAb 8644 20 µl
  • WB
  • IP
  • IF
  • ChIP
H 66 Rabbit IgG
Phospho-Estrogen Receptor α (Ser104/106) Antibody 2517 20 µl
  • WB
H 66 Rabbit 
Phospho-Estrogen Receptor α (Ser118) (16J4) Mouse mAb 2511 20 µl
  • WB
  • IHC
H 66 Mouse IgG2b
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

The Phospho-Estrogen Receptor α Antibody Sampler Kit provides an economical means to evaluate the activation status of ERα, including phosphorylation of Ser104/106, Ser167, and Ser118. The monoclonal control ERα antibody is also included to detect total Estrogen Receptor α levels. The kit contains enough primary antibody to perform four Western blot experiments.

Phospho-Estrogen Receptor α (Ser167) (D1A3) Rabbit mAb detects endogeneous levels of ERα protein only when phosphorylated at Ser167, and also cross reacts with a nonspecific band around 77 kDa. Phospho-Estrogen Receptor α (Ser104/106) Antibody detects endogenous levels of Ser104/106 phosphorylated ERα. Phospho-Estrogen Receptor α (Ser118) (16J4) Mouse mAb will detect endogenous levels of Ser118 phosphorylated ERα. Estrogen Receptor α (D8H8) Rabbit mAb detects endogenous levels of ERα. Each antibody in the kit does not cross-react with phosphorylated or nonphosphorylated ER isoforms β or other related family members.

Activation state monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides or non-phosphopeptide corresponding to residues surrounding Ser167, Ser118, and the carboxy terminus of human estrogen receptor α protein. Activation state polyclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser104/106 of human ERα. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Estrogen receptor α (ERα), a member of the steroid receptor superfamily, contains highly conserved DNA binding and ligand binding domains (1). Through its estrogen-independent and estrogen-dependent activation domains (AF-1 and AF-2, respectively), ERα regulates transcription by recruiting coactivator proteins and interacting with general transcriptional machinery (2). Phosphorylation at multiple sites provides an important mechanism to regulate ERα activity (3-5). Ser104, 106, 118, and 167 are located in the amino-terminal transcription activation function domain AF-1, and phosphorylation of these serine residues plays an important role in regulating ERα activity. Ser118 may be the substrate of the transcription regulatory kinase CDK7 (5). Ser167 may be phosphorylated by p90RSK and Akt (4,6). According to the research literature, phosphorylation at Ser167 may confer tamoxifen resistance in breast cancer patients (4).

  1. Mangelsdorf, D.J. et al. (1995) Cell 83, 835-9.
  2. Glass, C.K. and Rosenfeld, M.G. (2000) Genes Dev 14, 121-41.
  3. Chen, D. et al. (1999) Mol Cell Biol 19, 1002-15.
  4. Campbell, R.A. et al. (2001) J Biol Chem 276, 9817-24.
  5. Chen, D. et al. (2000) Mol Cell 6, 127-37.
  6. Joel, P.B. et al. (1998) Mol Cell Biol 18, 1978-84.
Entrez-Gene Id
2099
Swiss-Prot Acc.
P03372
For Research Use Only. Not For Use In Diagnostic Procedures.

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