Mouse, Human Predicted to work with:
Abcam’s SMAD3 (pS423/S425) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of SMAD3 (pS423/S425) protein in human and mouse cells.
The SimpleStep ELISA™ employs a labeled capture and detector antibody which immunocaptures the sample analyte in solution. This entire complex (capture antibody/protein/detector antibody) is in turn immobilized in the well by immunoaffinity via the anti-tag antibody. Samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material; the TMB substrate is then added. The reaction is stopped by addition of Stop Solution which stops the color development and completes any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
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.
Involvement in disease
Defects in SMAD3 may be a cause of colorectal cancer (CRC) [MIM:114500].
Belongs to the dwarfin/SMAD family. Contains 1 MH1 (MAD homology 1) domain. Contains 1 MH2 (MAD homology 2) domain.
The MH1 domain is required for DNA binding. Also binds zinc ions which are necessary for the DNA binding. The MH2 domain is required for both homomeric and heteromeric interactions and for transcriptional regulation. Sufficient for nuclear import. The linker region is required for the TGFbeta-mediated transcriptional activity and acts synergistically with the MH2 domain.
Phosphorylated on serine and threonine residues. Enhanced phosphorylation in the linker region on Thr-179, Ser-204 and Ser-208 on EGF AND TGF-beta treatment. Ser-208 is the main site of MAPK-mediated phosphorylation. CDK-mediated phosphorylation occurs in a cell-cycle dependent manner and inhibits both the transcriptional activity and antiproliferative functions of SMAD3. This phosphorylation is inhibited by flavopiridol. Maximum phosphorylation at the G(1)/S junction. Also phosphorylated on serine residues in the C-terminal SXS motif by TGFBR1 and ACVR1. TGFBR1-mediated phosphorylation at these C-terminal sites is required for interaction with SMAD4, nuclear location and transactivational activity, and appears to be a prerequisite for the TGF-beta mediated phosphorylation in the linker region. Dephosphorylated in the C-terminal SXS motif by PPM1A. This dephosphorylation disrupts the interaction with SMAD4, promotes nuclear export and terminates TGF-beta-mediated signaling. Acetylation in the nucleus by EP300 in the MH2 domain regulates positively its transcriptional activity and is enhanced by TGF-beta.
Cytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. Co-localizes with LEMD3 at the nucleus inner membrane. MAPK-mediated phosphorylation appears to have no effect on nuclear import.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Typical cell lysate dilution series
Example of a typical SMAD3 (pS423/S425) cell lysate dilution series. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed.
Linearity of dilution
Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1X Cell Extraction Buffer PTR. Data from duplicate measurements of SMAD3 (pS423/S425) are normalized and plotted.
SMAD3 (pS423/S425) phosphorylation in response to TGF-beta treatment.
Induction of SMAD3 (pS423/S425) phosphorylation in HeLa cells in response to TGF-β treatment. HeLa cells were cultured in 96-well tissue culture plates, serum-starved and treated (60 min) with a dose-range of TGF-β before cell lysis. Data from quadruplicate measurements of SMAD3 (pS423/S425) are plotted.
SMAD3 (pS423/S425) phosphorylation in response to SB431542 treatment.
Inhibition of SMAD3 (pS423/S425) phosphorylation in HeLa cells in response to SB431542 treatment. HeLa cells were cultured in 96-well tissue culture plates and treated with a dose-range of SB431542 (60 min). Cells were then stimulated with TGF-β (60 min) and lysed. Data from quadruplicate measurements of SMAD3 (pS423/S425) are plotted.