Western blot analysis of extracts from various cell lines using Smurf1 Antibody.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
Smurf1 Antibody detects endogenous levels of total Smurf1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a central region within human Smurf1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
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 Smad9 (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).
Smurf1, a member of the HECT family of E3 ubiquitin ligases, selectively interacts with BMP pathway Smad effectors, leading to Smad protein ubiquitination and degradation (6). In addition, Smurf1 interacts with the inhibitor Smad, Smad7, the bone-specific transcription factor Runx2/Cbfa1, RhoA and MEKK2 (7-10). Smurf1 negatively regulates osteoblast differentiation and bone formation in vivo (10,11). A related protein, Smurf2, acts more promiscuously, interacting with both BMP and TGF-β pathway Smad proteins (12).
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