Smad3 (Ser204) Polyclonal Antibody

$HUVEC cells(black) were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-Smad3 (Ser204) Polyclonal Antibody(bs-5235R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).$

HUVEC cells(black) were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-Smad3 (Ser204) Polyclonal Antibody(bs-5235R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

$Paraformaldehyde-fixed, paraffin embedded Rat uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:400 overnight at 4\u00b0C, DAB staining.$

Paraformaldehyde-fixed, paraffin embedded Rat uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:400 overnight at 4\u00b0C, DAB staining.

$Paraformaldehyde-fixed, paraffin embedded Mouse kidney; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.$

Paraformaldehyde-fixed, paraffin embedded Mouse kidney; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.

$Paraformaldehyde-fixed, paraffin embedded Rat kidney; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.$

Paraformaldehyde-fixed, paraffin embedded Rat kidney; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.

$Paraformaldehyde-fixed, paraffin embedded Human skin cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.$

Paraformaldehyde-fixed, paraffin embedded Human skin cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.

$Paraformaldehyde-fixed, paraffin embedded Human gastric carcinoma; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.$

Paraformaldehyde-fixed, paraffin embedded Human gastric carcinoma; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37\u00b0C for 30min; Antibody incubation with Smad3 (Ser204) Polyclonal Antibody, Unconjugated (bs-5235R) at 1:200 overnight at 4\u00b0C, DAB staining.

Lane 1: Mouse Heart lysates; Lane 2: Mouse Kidney lysates; Lane 3: Rat Skin lysates probed with Smad3 Polyclonal Antibody, Unconjugated (bs-5235R) at 1:1000 dilution and 4˚C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.

$A431 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Smad3 (Ser204) Polyclonal Antibody(bs-5235R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).$

A431 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Smad3 (Ser204) Polyclonal Antibody(bs-5235R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

$Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37\u00b0C for 20 min; Antibody incubation with (phospho-Smad3 (Ser204)) polyclonal Antibody, Unconjugated (bs-5235R) 1:100, 90 minutes at 37\u00b0C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37\u00b0C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.$

Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37\u00b0C for 20 min; Antibody incubation with (phospho-Smad3 (Ser204)) polyclonal Antibody, Unconjugated (bs-5235R) 1:100, 90 minutes at 37\u00b0C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37\u00b0C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Applications

WB
ELISA
FCM
IHC-P
IHC-F
IF(IHC-P)
IF(IHC-F)
IF(ICC)
ICC

Reactivity

Human
Mouse
Rat

Predicted Reactivity

Cow
Sheep
Pig
Horse
Rabbit

Overview
Catalog #	bs-5235R
Product Name	Smad3 (Ser204) Polyclonal Antibody
Applications	WB, ELISA, FCM, IHC-P, IHC-F, IF(IHC-P), IF(IHC-F), IF(ICC), ICC
Specificity	This phosphorylation site is homologous to that of Ser204 in Mouse and Rat.
Reactivity	Human, Mouse, Rat
Predicted Reactivity	Cow, Sheep, Pig, Horse, Rabbit
Specifications
Conjugation	Unconjugated
Host	Rabbit
Source	KLH conjugated synthetic phosphopeptide derived from human Smad3 around the phosphorylation site of Ser204 [AG(p-S)PN]
Modification Site	Ser204
Clonality	Polyclonal
Isotype	IgG
Concentration	1ug/ul
Purification	Purified by Protein A.
Storage Buffer	0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.
Storage Condition	Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.
Target
Gene ID	4088
Swiss Prot	P84022
Subcellular location	Cytoplasm, Nucleus
Synonyms	LDS3; LDS1C; MADH3; JV15-2; HSPC193; HsT17436; Mothers against decapentaplegic homolog 3; MAD homolog 3; Mad3; Mothers against DPP homolog 3; hMAD-3; SMAD family member 3; SMAD 3; Smad3; hSMAD3
Background	Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD3/SMAD4 complex, activates transcription. Also can form a SMAD3/SMAD4/JUN/FOS complex at the AP-1/SMAD site to regulate TGF-beta-mediated transcription. Has an inhibitory effect on wound healing probably by modulating both growth and migration of primary keratinocytes and by altering the TGF-mediated chemotaxis of monocytes. This effect on wound healing appears to be hormone-sensitive. Regulator of chondrogenesis and osteogenesis and inhibits early healing of bone fractures. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.
Application Dilution
WB	1:300-5000
ELISA	1:500-1000
FCM	1:20-100
IHC-P	1:200-400
IHC-F	1:100-500
IF(IHC-P)	1:50-200
IF(IHC-F)	1:50-200
IF(ICC)	1:50-200
ICC	1:100-500