Anti-Phospho-Smad3 (Ser425) Polyclonal Antibody is a Rabbit antibody targeting Phospho-Smad3 (Ser425). Anti-Phospho-Smad3 (Ser425) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.

IgG

Human,Mouse,Rat (predicted:Chicken,Pig,Cow,Horse,Rabbit)

1. Blank control (Black line): HUVEC (Black).
Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser425) antibody (TMAB-01500)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature.
2. Paraformaldehyde-fixed, paraffin embedded (Human colon cancer); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Phospho-Smad3 (Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01500) at 1:400 overnight at 4°C, followed by a conjugated secondary antibody for 20 min and DAB staining.
3. Tissue/cell: human colon carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37°C for 20 min;
Incubation: Anti-Phospho-Smad3 (Ser425) Polyclonal Antibody, Unconjugated (TMAB-01500) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
4. Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 μg
Lane 2: Heart (Mouse) Lysate at 40 μg
Lane 3: Kidney (Mouse) Lysate at 40 μg
Lane 4: Cerebrum (Rat) Lysate at 40 μg
Primary: Anti-Phospho-Smad3 (Ser425) (TMAB-01500) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kDa
Observed band size: 50 kDa
5. Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Phospho-Smad3 (Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01500) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
6. Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Phospho-Smad3 (Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01500) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 1μg/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human Smad3 around the phosphorylation site of Ser425

Human

SMAD3

Mothers against decapentaplegic homolog 3

Response to hypoxia,SMAD family,SMADs,Hypoxia,Cancer,SMADs,Cytoplasmic

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 (By similarity). Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.