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12807
Progesterone Receptor Signaling Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Progesterone Receptor Signaling Antibody Sampler Kit #12807

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Progesterone Receptor Signaling Antibody Sampler Kit: Image 1
Western blot analysis of extracts from T-47D cells grown for 48 hr in phenol red-free medium supplemented with 5% charcoal-stripped FBS, untreated (-) or promegestone (R5020)-treated (100 nM, 1 hr; +), using Phospho-Progesterone Receptor (Ser345) Antibody (upper) or Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb #8757 (lower).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 2
Western blot analysis of extracts from MCF-7 and T47D cells using Progesterone Receptor B (C1A2) Rabbit mAb.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 3
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Progesterone Receptor B Receptor (C1A2) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 4
Confocal immunofluorescent analysis of MCF-7 cells using Progesterone Receptor B Receptor (C1A2) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 5
Flow cytometric analysis of T-47D cells using Progesterone Receptor B Receptor (C1A2) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 6
Western blot analysis of extracts from T47D cells, untreated or stimulated with 100 nM promegestone (R5020) for 1 hour, using Phospho-Progesterone Receptor (Ser190) Antibody (upper) and control Progesterone Receptor Antibody #3172 (lower).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 7
Western blot analysis of extracts from COS cells, untreated or treated with either U0126 #9903 (10 µM for 1h) or TPA #4174 (200 nM for 10 m), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb #9107.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 8
Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA #4174 as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 9
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 10
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 11
Immunohistochemical analysis using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb on SignalSlide™ Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Controls #8103 (paraffin-embedded NIH/3T3 cells, treated with U0126 #9903 (left) or TPA #4174 (right).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 12
Confocal immunofluorescent analysis of Drosophila egg chambers, untreated (top) or λ phosphatase-treated (bottom), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 13
Confocal immunofluorescent analysis of HT1080 cells, starved overnight then treated with U0126 #9903 (10 uM, 2 h; left) or PDBu (Phorbol 12,13-Dibutyrate) #12808 (100 nM, 15 m; right) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 14
Flow cytometric analysis of Jurkat cells, treated with U0126 (10 µM, 2 hrs; green) or treated with TPA #4174 (200 nM, 30 min; blue) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 15
Western blot analysis of extracts NIH/3T3 cells, serum-starved or treated with human Platelet-Derived Growth Factor BB hPDGF-BB #8912 (100 ng/ml, 15 min), using Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb (upper) or Src (36D10) Rabbit mAb #2109 (lower).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 16
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.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 17
Chromatin immunoprecipitations were performed with cross-linked chromatin from T-47D cells cultured in phenol red-free media supplemented with 5% charcoal-stripped FBS for 48 hr and then treated with promegestone (R5020, 10 nM, 1 hr) and Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across FKBP5/FKBP51, a known target gene of Progesterone Receptor A/B (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 18
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).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 19
Chromatin immunoprecipitations were performed with cross-linked chromatin from T-47D cells cultured in phenol red-free media supplemented with 5% charcoal-stripped FBS for 48 hr and then promegestone-treated (R5020, 10 nM, 1 hr) and Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 6 (upper), including FKBP5/ FKBP51 (lower), a known target gene of Progesterone Receptor A/B (see additional figure containing ChIP-qPCR data).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 20
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.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 21
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 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.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 22
Immunohistochemical analysis of paraffin-embedded human infiltrating ductal breast carcinoma using Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 23
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.
Progesterone Receptor Signaling Antibody Sampler Kit: Image 24
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).
Progesterone Receptor Signaling Antibody Sampler Kit: Image 25
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.
Inquiry Info.# 12807

Product Description

Progesterone Receptor Signaling Antibody Sampler Kit provides an economical means of detecting total and active levels of progesterone receptor (PR) as well as the active forms of PR downstream targets. The kit contains enough primary antibody to perform four western blots per primary antibody.

Specificity / Sensitivity

Unless otherwise indicated, each antibody will recognize endogenous total levels of target protein. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb recognizes endogenous p44 and p42 MAP Kinase (Erk1/2) when dually phosphorylated at Thr202/Tyr204 of Erk1 (Thr185/Tyr187 of Erk2), and singly phosphorylated at Thr202. This antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinases. Phospho-Progesterone Receptor (Ser190) Antibody detects endogenous levels of progesterone receptors B and A only when phosphorylated at Ser190 and Ser26, respectively. Phospho-Progesterone Receptor (Ser345) Antibody recognizes endogenous levels of progesterone receptors B and A only when phosphorylated at Ser345 and Ser181, respectively. Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb may cross-react with other Src family proteins phosphorylated at equivalent sites or with overexpressed phosphorylated RTKs.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase, or with synthetic peptides corresponding to residues surrounding Ser115 or Tyr541 of human progesterone receptor protein, or Tyr416 of human Src protein. Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser190 of human progesterone receptor protein or Ser345 of human progesterone receptor B protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

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 (1,2). Both PR-A and PR-B are ligand activated, but differ in their relative ability to activate target gene transcription (3,4). The activity of PR is regulated by phosphorylation; at least seven serine residues are phosphorylated in its amino-terminal domain. Three sites (Ser81, Ser102, and Ser162) are unique to full length PR-B, while other sites (Ser190, Ser294, Ser345, and Ser400) are shared by both isoforms (5). Phosphorylation of PR-B at Ser190 (equivalent to Ser26 of PR-A) is catalyzed by CDK2 (6). Mutation of Ser190 results in decreased activity of PR (7), suggesting that the phosphorylation at Ser190 may be critical to its biological function. Research studies have demonstrated ligand-dependent phosphorylation of PR-B at Ser345 is catalyzed by MAPK and plays an important role in mediating the proliferation of breast cancer cells. Investigators have shown that Ser345-phosphorylated PR-B associates with Sp1 to regulate EGFR and p21 transcription (8). PR signaling has been shown to crosstalk with other kinase signaling cascades. Upon stimulation and the subsequent interaction with estrogen receptor α and c-Src, PR-B has been shown to promote the activation of the Src/p21ras/Erk pathway (9).
  1. Evans, R.M. (1988) Science 240, 889-95.
  2. Kastner, P. et al. (1990) EMBO J 9, 1603-14.
  3. Giangrande, P.H. et al. (2000) Mol Cell Biol 20, 3102-15.
  4. Wen, D.X. et al. (1994) Mol Cell Biol 14, 8356-64.
  5. Clemm, D.L. et al. (2000) Mol Endocrinol 14, 52-65.
  6. Zhang, Y. et al. (1997) Mol Endocrinol 11, 823-32.
  7. Takimoto, G.S. et al. (1996) J Biol Chem 271, 13308-16.
  8. Faivre, E.J. et al. (2008) Mol Endocrinol 22, 823-37.
  9. Blunt, R.J. et al. (1975) Pflugers Arch 355, 189-204.

Pathways & Proteins

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Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
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