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Store at –20°C
Phospho-Smad1/5 (Ser463/465)
(41D10) Rabbit mAb
#9516
n Small
100 µl
(10 western blots)
Orders n 877-616-CELL (2355)
[email protected]
Support n 877-678-TECH (8324)
[email protected]
Web n www.cellsignal.com
n Petite
40 µl
(4 western blots)
rev. 04/01/15
For Research Use Only. Not For Use In Diagnostic Procedures.
Entrez-Gene ID #4090
UniProt Acc. #Q99717
Applications
Species Cross-Reactivity*
Molecular Wt.
Isotype
W, IF-IC, F
Endogenous
H, M, R
60 kDa
Rabbit IgG**
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 is determined by western blot.
® 2015 Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Source/Purification: Monoclonal antibody is produced
by immunizing animals with a synthetic phosphopeptide
corresponding to residues surrounding Ser463/465 of human
Smad5.
kDa
HeLa
PhosphoSmad1/5
60
50
40
(3) Klemm, J.D. et al. (1998) Annu. Rev. Immunol. 16,
569-592.
(4) Kretzschmar, M. et al. (1997) Genes Dev. 11, 984-995.
Recommended Antibody Dilutions:
Western blotting
1:1000
Immunofluorescence (IF-IC)1:50
IF Protocol:
Methanol Permeabilization required
Flow Cytometry
1:400
For product specific protocols and a complete listing
of recommended companion products please see the
product web page at www.cellsignal.com
30
20
–
+
–
+
BMP-4
Western blot analysis of extracts from untreated or BMP4-treated HeLa or NIH/3T3 cells using Phospho-Smad1/5
(Ser463/465) (41D10) Rabbit mAb.
Tgf-b/Smads,Cytokine section
BMP sub-family ligands:
BMP2, -4, -7, MIS
type II
receptor
type I
receptor
type I
receptor
Smad2/3
HPK1
Thr183
P
P Tyr185
JNK
Erk
P
Smurf1
Smad1/5/8
Smad6
Thr202 Tyr204
P
Smad7
Smurf2 Erk
Smad2/3
TAK1
TAB1
Phospho-Smad1/5
type II
receptor
Ras
SARA
Flow cytometric analysis of HeLa cells, untreated (blue) or
BMP-treated (green), using Phospho-Smad1/5 (Ser463/465)
(41D10) Rabbit mAb compared to a nonspecific negative control
antibody (red).
Converted from
09_PW_Tgf-b_Smads_IHC_REV.eps
which uses different fonts
3/9/2004
TGF-β sub-family ligands:
TGF-βs, Activins, Nodals
Background References:
(1) Hogan, B.L. et al. (1996) Genes Dev. 10, 1580-1594.
(2) Hoodless, P.A. et al. (1996) Cell 85, 489-500.
**Anti-rabbit secondary antibodies must be used to
detect this antibody.
NIH/3T3
100
80
Events
Background: Bone morphogenetic proteins (BMPs)
constitute a large family of signaling molecules that regulate
a wide range of critical processes including morphogenesis,
cell-fate determination, proliferation, differentiation, and
apoptosis (1,2). BMP receptors are members of the TGF-β
family of Ser/Thr kinase receptors. Ligand binding induces
multimerization, autophosphorylation, and activation of
these receptors (3-5). They subsequently phosphorylate
Smad1 at Ser463 and Ser465 in the carboxy-terminal motif
SSXS, as well as Smad5 and Smad8 at their corresponding sites. These phosphorylated Smads dimerize with the
coactivating Smad4 and translocate to the nucleus, where
they stimulate transcription of target genes (5).
MAP kinases and CDKs 8 and 9 phosphorylate residues in
the linker region of Smad1, including Ser206. The phosphorylation of Ser206 recruits Smurf1 to the linker region
and leads to the degradation of Smad1 (6). Phosphorylation
of this site also promotes Smad1 transcriptional action by
recruiting YAP to the linker region (7).
Specificity/Sensitivity: Phospho-Smad1/5 (Ser463/465)
(41D10) Rabbit mAb detects endogenous levels of Smad1
and Smad5 only when dually phosphorylated at Ser463 and
Ser465 and is also predicted to detect Smad9 (Smad8) when
phosphorylated at Ser465 and Ser467. The antibody does not
cross-react with other Smad-related proteins.
Smad4
(common)
Ser465
P
Ser467
Smad2/3 P
Smad4
Smad1/5/8
Smad4
TGIF
(5) Whitman, M. (1998) Genes Dev. 12, 2445-2462.
CBP Smad1/5/8
Smad4
TF
CBP
Smad2/3
Smad4
TF
(6) Sapkota, G. et al. (2007) Mol Cell 25, 441-54.
(7) Alarcón, C. et al. (2009) Cell 139, 757-69.
IMPORTANT: For western blots, incubate membrane
with diluted antibody in 5% w/v BSA, 1X TBS, 0.1%
Tween® 20 at 4°C with gentle shaking, overnight.
U.S. Patent No. 5,675,063
DyLight is a trademark of Thermo Fisher Scientific Inc. and its
subsidiaries.
Tween is a registered trademark of ICI Americas, Inc.
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
All—all species expected
Mi—mink
C—chicken
IF—Immunofluorescence
F—Flow cytometry
Dm—D. melanogaster X—Xenopus
Z—zebrafish
Species enclosed in parentheses are predicted to react based on 100% homology.
E-P—ELISA-Peptide
B—bovine
Serum-starved
hBMP2-treated
® 2015 Cell Signaling Technology, Inc.
Confocal immunofluorescent analysis of HT1080 cells, serum-starved (left) or serum-starved then treated
with hBMP2 (50 ng/ml, 30 min; right) using Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb (green)
and Cox IV (4D11-B3-E8) Mouse mAb #11967 (red).
Orders n 877-616-CELL (2355)
[email protected]
Support n 877-678-TECH (8324)
[email protected]
Web n www.cellsignal.com