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8595
Nuclear Receptor Antibody Sampler Kit

Nuclear Receptor Antibody Sampler Kit #8595

 Image 1

Western blot analysis of extracts from NIH/3T3 and 3T3-L1 cells (differentiated 6 d) using PPARγ (C26H12) Rabbit mAb #2435.

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 Image 2

Western blot analysis of extracts from NIH/3T3 and C6 cells using RARα Antibody #2554.

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 Image 3

Western blot analysis of extracts from various cell lines using RXRα (D6H10) Rabbit mAb #3085.

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 Image 4

Western blot analysis of extracts from AR-positive (LNCaP and MCF7) and AR-negative (PC-3 and DU 145) cell lines using Androgen Receptor (D6F11) XP® Rabbit mAb #5153 (upper) or β-Actin Antibody #4967 (lower).

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 Image 5

Western blot analysis of extracts from ER-positive (MCF7, T-47D, ZR-75-1) and ER-negative (SK-BR-3 and MCF 10A) cell lines using Estrogen Receptor α (D8H8) Rabbit mAb #8644 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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 Image 6

Western blot analysis of extracts from PR-positive (T-47D) and PR-negative (MDA-MB-231) cell lines using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb #8757 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

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 Image 7

Western blot analysis of extracts from various cell lines using RARγ1 (D3A4) XP® Rabbit mAb #8965.

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Western Blotting Image 8

Western blot analysis of extracts from NIH/3T3 and C6 cells, using RARα Antibody.

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Western Blotting Image 9

Western blot analysis of extracts from various cell lines using RARγ1 (D3A4) XP® Rabbit mAb.

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Western Blotting Image 10

Western blot analysis of extracts from various cell lines using RXRα (D6H10) Rabbit mAb.

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Western Blotting Image 11

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human glucocorticoid receptor-α (hGRα-Myc/DDK, +) or Myc/DDK-tagged full-length human mineralocorticoid receptor (hMR-Myc/DDK, +), using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (upper) and DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower).

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Western Blotting Image 12

Western blot analysis of extracts from T-47D (PR positive) and MDA-MB-231 (PR negative) cells using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

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Western Blotting Image 13

Western blot analysis of extracts from LNCaP (AR+), MCF7 (AR+), PC-3 (AR-), and DU 145 (AR-) cells using Androgen Receptor (D6F11) XP® Rabbit mAb (upper) and β-Actin Antibody #4967 (lower).

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Western Blotting Image 14

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 15

Chromatin immunoprecipitations were performed with cross-linked chromatin from 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 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 16

Western blot analysis of extracts from NIH/3T3 and 3T3-L1 cells (differentiated 6 days) using PPARγ (C26H12) Rabbit mAb.

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Western Blotting Image 17

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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Western Blotting Image 18

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human RARγ1 (hRARγ1-Myc/DDK, +), using RARγ1 (D3A4) XP® Rabbit mAb.

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Isoform Specificity Image 19

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with Myc/DDK-tagged cDNA expression constructs encoding full-length human RXRα (hRXRα; +), RXRβ (hRXRβ; +), or RXRγ (hRXRγ; +), using RXRα (D6H10) Rabbit mAb (upper) and DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower).

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Western Blotting Image 20

Western blot analysis of extracts from various cell lines using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb.

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Western Blotting Image 21

Western blot analysis of extracts from T-47D cells, grown for 48 hr in phenol red-free medium supplemented with 5% charcoal-stripped FBS and then treated with either a vehicle control (-) or promegestone (R5020, 100 nM, 16 hr; +), using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Prolonged treatment of PR-expressing cells with R5020 is known to induce PR downregulation and hyperphosphorylation, which is reflected by slower migration on SDS-PAGE.

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Chromatin IP Image 22

T-47D cells were cultured in phenol red-free media supplemented with 5% charcoal-stripped FBS for 48 hr and then either untreated (left panel) or promegestone-treated (R5020, 10 nM, 1 hr; right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin cells and Progesterone Receptor A/B (D8Q2J) XP® 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 FKBP51 Intron 5 Primers #7859, human E2F-1 proximal enhancer site #1 primers, 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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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded LNCaP (AR+, left) and DU145 (AR-, right) using Androgen Receptor (D6F11) XP® Rabbit mAb.

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IF-IC Image 24

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 25

Chromatin immunoprecipitations were performed with cross-linked chromatin from 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 Estrogen Receptor α (D8H8) Rabbit mAb or 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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IHC-P (paraffin) Image 26

Immunohistochemical analysis of 3T3-L1 cells, undifferentiated (left) or differentiated (right) , using PPARγ (C26H12) Rabbit mAb.

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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded cell pellets, HaCaT (positive, left) and Hep3B (negative, right), using RARγ1 (D3A4) XP® Rabbit mAb.

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Flow Cytometry Image 28

Human whole blood was fixed, lysed, and permeabilized as per the Cell Signaling Technology Flow Alternate Protocol and stained using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. Samples were gated on CD3+ lymphocytes.

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IHC-P (paraffin) Image 29

Immunohistochemical analysis of paraffin-embedded human infiltrating ductal breast carcinoma using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb.

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IHC-P (paraffin) Image 30

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Androgen Receptor (D6F11) XP® Rabbit mAb.

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IHC-P (paraffin) Image 31

Immunohistochemical analysis of paraffin-embedded mouse brown fat using PPARγ (C26H12) Rabbit mAb.

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IHC-P (paraffin) Image 32

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RARγ1 (D3A4) XP® Rabbit mAb.

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IF-IC Image 33

Confocal immunofluorescent analysis of HeLa cells, grown in phenol red-free media containing 5% charcoal-stripped FBS for 2 days and either untreated (left) or dexamethasone-treated (100 nM, 2hr; right), using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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IHC-P (paraffin) Image 34

Immunohistochemical analysis of paraffin-embedded cell pellets, T-47D (high PR, left), MCF-7 (low PR, middle) and MDA-MB-231 (PR negative, right), using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb.

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Flow Cytometry Image 35

Flow cytometric analysis of DU-145 cells (red) and LNCaP cells (blue) using Androgen Receptor (D6F11) XP® Rabbit mAb.

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IF-IC Image 36

Confocal immunofluorescent analysis of 3T3-L1 cells using PPARγ (C26H12A8) Rabbit mAb (red) showing nuclear localization in differentiated cells. Lipid droplets have been labeled with BODIPY 493/503 (green). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

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IHC-P (paraffin) Image 37

Immunohistochemical analysis of paraffin-embedded human skin using RARγ1 (D3A4) XP® Rabbit mAb.

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Chromatin IP Image 38

A549 cells were cultured in media with 5% charcoal-stripped FBS for 3 days and then either untreated (left panel) or dexamethasone-treated (100 nM, 1 hr; right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin and Glucocorticoid Receptor (D8H2) XP® Rabbit mAb or 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 SLC19A2 Promoter Primers #7681, human MT2A promoter primers, 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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Flow Cytometry Image 39

Flow cytometric analysis of MDA MB-231 cells (blue) and T47D cells using Progesterone REceptor A/B (D8Q2J) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (ALexa FLuor® 488 Conjugate) #4412 was used as a secondary antibody.

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IF-IC Image 40

Confocal immunofluorescent analysis of LNCaP (positive, left) and DU145 (negative, right) cells using Androgen Receptor (D6F11) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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IF-IC Image 41

Confocal immunofluorescent analysis of HaCaT cells (positive, left) and Hep3B cells (negative, right) using RARγ1 (D3A4) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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IF-IC Image 42

Confocal immunofluorescent analysis of T-47D (PR positive, left) and MDA-MB-231 (PR negative, right) cells using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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Chromatin IP Image 43

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 LNCaP cells grown in phenol red free medium and 5% charcoal stripped FBS for 3 d then treated with dihydrotestosterone (DHT, 10 nM) for 4 hours and either Androgen Receptor (D6F11) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human KLK2 Intron 1 Primers #62086, SimpleChIP® Human KLK3 Promoter Primers #32784, 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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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
RARα Antibody 2554 20 µl
  • WB
M R 55 Rabbit 
RARγ1 (D3A4) XP® Rabbit mAb 8965 20 µl
  • WB
  • IP
  • IHC
  • IF
H M 58 Rabbit IgG
RXRα (D6H10) Rabbit mAb 3085 20 µl
  • WB
  • IP
H M R 53 Rabbit IgG
Glucocorticoid Receptor (D8H2) XP® Rabbit mAb 3660 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R Mk 80, 91, 94 Rabbit IgG
Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 8757 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H 90 (PR-A), 118 (PR-B) Rabbit IgG
Androgen Receptor (D6F11) XP® Rabbit mAb 5153 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H 110 Rabbit IgG
Estrogen Receptor α (D8H8) Rabbit mAb 8644 20 µl
  • WB
  • IP
  • IF
  • ChIP
H 66 Rabbit IgG
PPARγ (C26H12) Rabbit mAb 2435 20 µl
  • WB
  • IHC
  • IF
H M 53, 57 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Nuclear Receptor Antibody Sampler Kit provides an economical means to evaluate the presence and status of nuclear receptors. This kit contains enough primary antibody to perform two western blots per primary.

Each antibody in the Nuclear Receptor Antibody Sampler Kit recognizes endogenous levels of total respective protein. Glucocorticoid Receptor (D8H2) XP® Rabbit mAb is predicted to cross-react with all known alternative translation start site generated isoforms of glucocorticoid receptor-α and glucocorticoid receptor-β, and does not cross-react with mineralocorticoid receptor. Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb does not cross-react with either the glucocorticoid receptor or the mineralocorticoid receptor. RARγ1 (D3A4) XP® Rabbit mAb is not predicted to cross-react with RARγ2, and does not cross-react with either RARα or RARβ. RXRα (D6H10) Rabbit mAb does not cross-react with either RXRβ or RXRγ.

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human RARα protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminal region of human androgen receptor protein, residues in the carboxy terminus of human ERα protein, residues surrounding Leu378 of human glucocorticoid receptor protein, residues surrounding Asp69 of human PPARγ protein, residues surrounding Tyr541 of human progesterone receptor protein, residues near the amino terminus of human RARγ1 protein, or residues near the amino terminus of human RXRα protein.

Nuclear Receptors are transcription factors responsible for sensing bioactive molecules, including steroid and thyroid hormones. They are regulated by multiple posttranslational modifications, which in turn impacts their ability to regulate the expression of specific genes involved in the control of reproduction, development, and metabolism.

Androgen receptor (AR), a zinc finger transcription factor belonging to the nuclear receptor superfamily, is activated by phosphorylation and dimerization upon ligand binding (1). This promotes nuclear localization and binding of AR to androgen response elements in androgen target genes. AR plays a crucial role in several stages of male development and the progression of prostate cancer (2,3).

Estrogen receptor α (ERα), a member of the steroid receptor superfamily, contains highly conserved DNA binding and ligand binding domains (4). 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 (5).

Glucocorticoid hormones control cellular proliferation, inflammation, and metabolism through their association with the glucocorticoid receptor (GR)/NR3C1, a member of the nuclear hormone receptor superfamily of transcription factors (6).

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the ligand-activated nuclear receptor superfamily and functions as a transcriptional activator (7). PPARγ is preferentially expressed in adipocytes, as well as in vascular smooth muscle cells and macrophages (8). Besides its role in mediating adipogenesis and lipid metabolism (8), PPARγ also modulates insulin sensitivity, cell proliferation, and inflammation (9).

Human progesterone receptor (PR) is expressed as two forms: the full length PR B and the short form PR A. PR A lacks the first 164 amino acid residues of PR B (10,11). Both PR A and PR B are ligand activated, but differ in their relative ability to activate target gene transcription (12,13).

Nuclear retinoic acid receptors (RARs) consist of three subtypes encoded by separate genes: α (NR1B1), β (NR1B2), and γ (NR1B3). For each subtype, there are at least two isoforms, which are generated by differential promoter usage and alternative splicing and differ only in their N-terminal regions. Retinoids, which are metabolites of vitamin A, serve as ligands for RARs (14). RARs function as ligand-dependent transcriptional regulators and are found to be heterodimerized with retinoid X receptors (RXRs). These transcriptionally active dimers regulate the expression of genes involved in cellular differentiation, proliferation, and apoptosis (15,16).

The human retinoid X receptors are encoded by three distinct genes (RXRα, RXRβ, and RXRγ) and bind selectively and with high affinity to the vitamin A derivative, 9-cis-retinoic acid. RXRs are type-II nuclear hormone receptors that are largely localized to the nuclear compartment independent of ligand binding. Nuclear RXRs form heterodimers with nuclear hormone receptor subfamily 1 proteins, including thyroid hormone receptor, retinoic acid receptors, vitamin D receptor, peroxisome proliferator-activated receptors, liver X receptors, and farnesoid X receptor (17).

  1. Li, J. and Al-Azzawi, F. (2009) Maturitas 63, 142-8.
  2. Evans, R.M. (1988) Science 240, 889-895.
  3. Avila, D.M. et al. (2001) J. Steroid. Biochem. Mol. Biol. 76, 135-142.
  4. Kastner, P. et al. (1990) EMBO J. 112, 1603-1614.
  5. Montgomery, J.S. et al. (2001) J. Pathol. 195, 138-146.
  6. Giangrande, P.H. et al. (2000) Mol. Cell. Biol. 20, 3102-3115.
  7. Mangelsdorf, D.J. et al. (1995) Cell 83, 835-9.
  8. Wen, D.X. et al. (1994) Mol. Cell. Biol. 14, 8356-8364.
  9. Glass, C.K. and Rosenfeld, M.G. (2000) Genes Dev 14, 121-41.
  10. Yamamoto, K.R. (1985) Annu. Rev. Genet 19, 209-252.
  11. Tontonoz, P. et al. (1995) Curr Opin Genet Dev 5, 571-6.
  12. Rosen, E.D. et al. (1999) Mol Cell 4, 611-7.
  13. Murphy, G.J. and Holder, J.C. (2000) Trends Pharmacol Sci 21, 469-74.
  14. Rochette-Egly, C. and Germain, P. (2009) Nucl Recept Signal 7, e005.
  15. Delacroix, L. et al. (2010) Mol Cell Biol 30, 231-44.
  16. Eifert, C. et al. (2006) Mol Reprod Dev 73, 796-824.
  17. Gronemeyer, H. et al. (2004) Nat Rev Drug Discov 3, 950-64.
Entrez-Gene Id
367 , 2099 , 2908 , 5468 , 5241 , 5914 , 5916 , 6256
Swiss-Prot Acc.
P10275 , P03372 , P04150 , P37231 , P06401 , P10276 , P13631 , P19793
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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