Store at -20°C
Progesterone Receptor A/B (D8Q2J)
XP® Rabbit mAb
#8757
Orders n 877-616-CELL (2355)
[email protected]
Support n 877-678-TECH (8324)
[email protected]
Web n www.cellsignal.com
rev. 03/07/16
For Research Use Only. Not For Use In Diagnostic Procedures.
Entrez-Gene ID #5241
UniProt ID #P06401
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.
Species Cross-Reactivity*
Isotype
Applications
Molecular Wt.
W, IP, IHC-P, IF-IC, ChIP, F
H, (Mk)
90 (PR-A), 118 (PR-B) kDa Rabbit IgG**
Endogenous
*Species cross-reactivity is determined by western blot.
® 2015 Cell Signaling Technology, Inc.
XP, SignalStain, SimpleChIP and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc.
Specificity/Sensitivity: Progesterone Receptor A/B
(D8Q2J) XP® Rabbit mAb recognizes endogenous levels of
total progesterone receptor A and B proteins. This antibody
does not cross-react with either the glucocorticoid receptor
or the mineralocorticoid receptor.
Source/Purification: Monoclonal antibody is produced
by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr541 of human progesterone
receptor protein.
**Anti-rabbit secondary antibodies must be used to
detect this antibody.
Recommended Antibody Dilutions:
Western blotting
1:1000
Immunoprecipitation1:50
Immunohistochemistry (Paraffin)
1:1000†
Unmasking buffer: Citrate
Antibody diluent: SignalStain® Antibody Diluent #8112
Detection reagent: SignalStain® Boost (HRP, Rabbit) #8114
†Optimal IHC dilutions determined using SignalStain® Boost IHC
Detection Reagent.
Immunofluorescence (IF-IC)1:800
Chromatin IP
1:100
Flow Cytometry
1:200
M
T4
kDa
7D
DA
-M
B-
23
1
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.
200
140
PR-B
100
PR-A
80
60
50
40
30
For product specific protocols and a complete listing
of recommended companion products please see the
product web page at www.cellsignal.com
40
GAPDH
30
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).
kDa
200
140
PR-B
100
PR-A
80
60
Western blot analysis of extracts from T-47D cells, grown for 48
hr in phenol red-free medium supplemented with 5% charcoalstripped 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.
50
Immunohistochemical analysis of paraffin-embedded human
infiltrating ductal breast carcinoma using Progesterone Receptor
A/B (D8Q2J) XP® Rabbit mAb.
40
30
40
GAPDH
30
–
+
R5020
IMPORTANT: For western blots, incubate membrane with diluted antibody in 5% w/v nonfat dry
milk, 1X TBS, 0.1% Tween®20 at 4°C with gentle shaking, overnight.
Applications Key:
W—Western
Species Cross-Reactivity Key:
IP—Immunoprecipitation
H—human
M—mouse
Dg—dog Pg—pig Sc—S. cerevisiae Ce—C. elegans
IHC—Immunohistochemistry
R—rat
Hr—horse
Hm—hamster
ChIP—Chromatin Immunoprecipitation
Mk—monkey
Mi—mink
All—all species expected
C—chicken
DyLight is a trademark of Thermo Fisher Scientific Inc. and its
subsidiaries.
Tween is a registered trademark of ICI Americas, Inc.
IF—Immunofluorescence
F—Flow cytometry
Dm—D. melanogaster X—Xenopus
Z—zebrafish
E-P—ELISA-Peptide
B—bovine
Species enclosed in parentheses are predicted to react based on 100% homology.
Immunohistochemical analysis of paraffin-embedded cell pellets, T-47D (high PR, left), MCF7 (low PR, middle), and MDA-MB-231 (PR negative, right), using Progesterone Receptor A/B (D8Q2J)
XP® Rabbit mAb.
Background References:
(1) Evans, R.M. (1988) Science 240, 889-895.
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0
Signal relative to input
Signal relative to input
Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb #8757
Normal Rabbit IgG #2729
FKBP51
E2F-1
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0
α Satellite
(2) Kastner, P. et al. (1990) EMBO J. 112, 1603-1614.
(3) Giangrande, P.H. et al. (2000) Mol. Cell. Biol. 20,
3102-3115.
(4) Wen, D.X. et al. (1994) Mol. Cell. Biol. 14, 8356-8364.
(5) Clemm, D.L. et al. (2000) Mol. Endocrinol. 14, 52-65.
FKBP51
E2F-1
(6) Zhang, Y. et al. (1997) Mol. Endocrinol. 11, 823-832.
α Satellite
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 from 4 x 106 cells and 5 µl of 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.
MDA-MB-231
Events
T-47D
(7) Takimoto, G.S. et al. (1996) J. Biol. Chem. 271,
13308-13316.
® 2015 Cell Signaling Technology, Inc.
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 DyLight™ 554 Phalloidin #13054 (red).
Orders n 877-616-CELL (2355)
[email protected]
Progesterone Receptor A/B
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.
Support n 877-678-TECH (8324)
[email protected]
Web n www.cellsignal.com