Revision 4
#61949
Store at -20C
Androgen Receptor Antibody Sampler Kit
1 Kit
(3 x 20 microliters)
Orders:
877-616-CELL (2355)
Support:
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
| Product Includes | Product # | Quantity | Mol. Wt | Isotype/Source |
|---|---|---|---|---|
| Androgen Receptor (E3S4N) Rabbit mAb (Carboxy-terminal Antigen) | 70317 | 20 µl | 110 kDa | Rabbit IgG |
| Androgen Receptor (D6F11) XP® Rabbit mAb | 5153 | 20 µl | 110 kDa | Rabbit IgG |
| Androgen Receptor (AR-V7 Specific) (E3O8L) Rabbit mAb | 19672 | 20 µl | 80 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
The Androgen Receptor Antibody Sampler Kit provides an economical means of detecting full-length Androgen Receptor and AR-V7 isoforms. The kit includes enough antibody to perform two western blot experiments with each primary antibody.
Background
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. Research studies have shown that AR plays a crucial role in several stages of male development and the progression of prostate cancer (2,3).
The AR3 or AR-V7 isoform, which lacks the typical ligand binding domain, is created through the alternative splicing of cryptic exons (4-5). AR-V7 is frequently expressed in castration-resistant prostate cancer (CRPC) and while dependent on the activity of the full-length androgen receptor (AR-FL), AR-V7 can activate a completely distinct transcriptional program (6-8). While enzalutamide and abiraterone have been beneficial in treating CRPC through the ligand binding domain of AR-FL, resistance in patients has been shown to be associated with AR-V7 detection in circulating tumor cells (9-12). Studies probing into mechanisms of overcoming this resistance are currently being explored and may help in stratifying patient populations for more personalized therapies (13-15).
The AR3 or AR-V7 isoform, which lacks the typical ligand binding domain, is created through the alternative splicing of cryptic exons (4-5). AR-V7 is frequently expressed in castration-resistant prostate cancer (CRPC) and while dependent on the activity of the full-length androgen receptor (AR-FL), AR-V7 can activate a completely distinct transcriptional program (6-8). While enzalutamide and abiraterone have been beneficial in treating CRPC through the ligand binding domain of AR-FL, resistance in patients has been shown to be associated with AR-V7 detection in circulating tumor cells (9-12). Studies probing into mechanisms of overcoming this resistance are currently being explored and may help in stratifying patient populations for more personalized therapies (13-15).
Background References
- Li, J. and Al-Azzawi, F. (2009) Maturitas 63, 142-8.
- Avila, D.M. et al. J Steroid Biochem Mol Biol 76, 135-42.
- Montgomery, J.S. et al. (2001) J Pathol 195, 138-46.
- Hu, R. et al. (2009) Cancer Res 69, 16-22.
- Guo, Z. et al. (2009) Cancer Res 69, 2305-13.
- Watson, P.A. et al. (2010) Proc Natl Acad Sci U S A 107, 16759-65.
- Sun, S. et al. (2010) J Clin Invest 120, 2715-30.
- Hu, R. et al. (2012) Cancer Res 72, 3457-62.
- Scher, H.I. et al. (2012) N Engl J Med 367, 1187-97.
- de Bono, J.S. et al. (2011) N Engl J Med 364, 1995-2005.
- Ryan, C.J. et al. (2013) N Engl J Med 368, 138-48.
- Antonarakis, E.S. et al. (2014) N Engl J Med 371, 1028-38.
- Liu, C. et al. (2014) Clin Cancer Res 20, 3198-3210.
- Sarwar, M. et al. (2016) Oncotarget 7, 63065-63081.
- Ku, S.Y. et al. (2017) Science 355, 78-83.
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
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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.
Revision 4
Western blot analysis of extracts from various cell lines using Androgen Receptor (AR-V7 Specific) (E3O8L) Rabbit mAb (upper) and Β-Actin (D6A8) Rabbit mAb #8457 (lower). Signal corresponding to the androgen receptor V7 isoform is detected in 22Rv1 and VCAP cells (AR-V7-positive), but is not detected in LNCaP and DU 145 cells (AR-V7-negative), confirming specificity of the antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 22-RV-1 cells, treated with 10 nM DHT for 4hr and either Androgen Receptor (AR-V7 Specific) (E3O8L) 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 SPINK5 upstream primers, ABCB5 upstream 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.
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).
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 4
CUT&RUN was performed with 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 Androgen Receptor (D6F11) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across KLK2, a known target gene of Androgen Receptor (see additional figure containing CUT&RUN-qPCR data).
Western blot analysis of extracts from various cell lines using Androgen Receptor (E3S4N) Rabbit mAb (Carboxy-terminal Antigen) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, DU 145 cells are negative for Androgen Receptor expression.
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.
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 4
Immunoprecipitation of AR-V7 from 22Rv1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb XP® Isotype Control #3900, and lane 3 is Androgen Receptor (AR-V7 Specific) (E3O8L) Rabbit mAb. Western blot analysis was performed using Androgen Receptor (AR-V7) (E3O8L) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded LNCaP (AR+, left) and DU145 (AR-, right) using Androgen Receptor (D6F11) XP® Rabbit mAb.
CUT&RUN was performed with 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 Androgen Receptor (D6F11) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 19 (upper), including KLK2 (lower), a known target gene of Androgen Receptor (see additional figure containing CUT&RUN-qPCR data).
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 4
Immunoprecipitation of Androgen Receptor from LNCaP cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Androgen Receptor (E3S4N) Rabbit mAb (Carboxy-terminal Antigen). Western blot analysis was performed using Androgen Receptor (E3S4N) Rabbit mAb (Carboxy-terminal Antigen). Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Androgen Receptor (D6F11) XP® Rabbit mAb.
CUT&RUN was performed with 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 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 SimpleChIP® Human KLK2 Intron 1 Primers #62086 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.
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 4
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).
Flow cytometric analysis of DU-145 cells (blue) and LNCaP cells (green) using Androgen Receptor (D6F11) XP® Rabbit mAb (solid lines) or a 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.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 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 Androgen Receptor (D6F11) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across KLK2, a known target gene of Androgen Receptor (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
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 4
Chromatin immunoprecipitations were performed with cross-linked chromatin from 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 Androgen Receptor (D6F11) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 19 (upper), including KLK2 (lower), a known target gene of Androgen Receptor (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from 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.
Western blot analysis of extracts from LNCaP (AR+), VCaP (AR & AR-V7+), 22Rv1 (AR & AR-V7+), and PC-3 (AR-) cells using Androgen Receptor (D6F11) XP® Rabbit mAb (upper), and β-Actin (13E5) Rabbit mAb (lower).
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 4
Simple Western™ analysis of lysates (0.1 mg/mL) from LNCaP cells using Androgen Receptor (D6F11) XP® Rabbit mAb #5153. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (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.
Confocal immunofluorescent analysis of LNCaP cells (left, positive) and DU 145 cells (right, negative) usingAndrogen Receptor (E3S4N) Rabbit mAb (Carboxy-terminal Antigen) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
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.