Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb 4370 40 µl 44, 42 kDa Rabbit IgG
Phospho-Progesterone Receptor (Ser190) Antibody 3171 40 µl 90, 118 kDa Rabbit 
Phospho-Progesterone Receptor (Ser345) Antibody 12783 40 µl 90 (PR-A), 118 (PR-B) kDa Rabbit 
Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb 8757 40 µl 90 (PR-A), 118 (PR-B) kDa Rabbit IgG
Progesterone Receptor B (C1A2) Rabbit mAb 3157 40 µl 118 kDa Rabbit 
Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb 6943 40 µl 60 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #12807

    Progesterone Receptor Signaling Antibody Sampler Kit

    Progesterone Receptor Signaling Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved HeLa cells treated with TPA (400 nM, 4 hours) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Progesterone Receptor Signaling Antibody Sampler Kit: Image 2 Expand Image
    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 3 Expand Image
    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 4 Expand Image
    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 5 Expand Image
    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 6 Expand Image
    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 7 Expand Image
    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 8 Expand Image
    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 9 Expand Image
    CUT&RUN was performed with 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 CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across SETD9 gene.
    Progesterone Receptor Signaling Antibody Sampler Kit: Image 10 Expand Image
    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 11 Expand Image
    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 12 Expand Image
    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 13 Expand Image
    CUT&RUN was performed with 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 CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 5 (upper), including SETD9 (lower).
    Progesterone Receptor Signaling Antibody Sampler Kit: Image 14 Expand Image
    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 15 Expand Image
    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 16 Expand Image
    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 17 Expand Image
    CUT&RUN was performed with 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 either Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human TBL2 promoter primers and human OCM2 exon 1 primers. 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 18 Expand Image
    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 19 Expand Image
    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 20 Expand Image
    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 21 Expand Image
    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 22 Expand Image
    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 23 Expand Image
    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 24 Expand Image
    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 25 Expand Image
    Flow cytometric analysis of MDA-MB-231 cells (blue, negative) and T47D cells (green, positive) using Progesterone Receptor A/B (D8Q2J) 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 26 Expand Image
    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 27 Expand Image
    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 Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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 datasheet.
    Progesterone Receptor Signaling Antibody Sampler Kit: Image 28 Expand Image
    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 Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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 29 Expand Image
    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 30 Expand Image
    Immunoprecipitation of Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) from 3T3 cell extracts. Cells were treated with TPA, (200 nM, 15 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb. Western blot was performed using Phosphop44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) mAb #45262 was used as a secondary antibody.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.