Catalog # NP4031

eNOS (Tyr-657)/nNOS (Tyr-895), phospho-specific Antibody

Rabbit Polyclonal

Application Dilution
ELISA1:2000
ICC1:400
WB1:1000

Size 100 μl

Species Reactivity Hu, Rt, Ms

MW 140 kDa

DATASHEET  




$295

Nitric oxide (NO) has a broad range of biological activities and is implicated in signaling pathways in phylogenetically diverse species. Nitric oxide synthases (NOS), the enzymes responsible for synthesis of NO, are homodimers whose monomers are themselves two fused enzymes: a cytochrome reductase and a cytochrome that requires three cosubstrates (L-arginine, NADPH, and oxygen) and five cofactors or prosthetic groups (FAD, FMN, calmodulin, tetrahydrobiopterin, and heme). Several distinct NOS isoforms are produced from three distinct genes, inducible NOS (iNOS, NOS-II), neuronal NOS (bNOS, NOS-I), and endothelial NOS (eNOS, ecNOS, NOS-III). Regulation of eNOS activity occurs through phosphorylation at multiple sites. Phosphorylation of Ser-633 (mouse Ser-632) in the FMN binding domain increases eNOS activity and may be important for the maintenance of NO synthesis after initial activation by Ca2+ flux and Ser-1177 phosphorylation. Tyr-657 is phosphorylated by PYK2 in response to fluid shear stress and this phosphorylation leads to attenuation of enzyme activity.

 

References

Loot, A. E. et al. (2009) J Exp Med. 206(13):2889.
Fisslthaler, B. et al. (2008) Circ Res. 102:1520.
Mount, P.F. et al. (2007) J Mo.l Cell. Cardiol. 42(2):271.
Musicki, B. et al. (2005) Proc. Natl. Acad. Sci.102(33):11870.

Western blot analysis of human umbilical vein endothelial cells stimulated with pervanadate (1 mM) for 30 min. (lanes 1, 3, & 5) then the blot was treated with alkaline phosphatase (lanes 2, 4, & 6). The blots were probed with anti-eNOS monoclonal antibody (NM2211; lanes 1 & 2), anti-eNOS (Tyr-657) phospho-specific antibody (NP4031; lanes 3 & 4), or anti-eNOS polyclonal antibody (NP2281; lanes 5 & 6).

Immunocytochemical labeling of nNOS phosphorylation in rat PC12 cells differentiated with NGF. The cells were probed with mouse monoclonal (mAb) nNOS (NM4011), and rabbit polyclonal (pAb) nNOS (C-terminal region), nNOS (Tyr-895)/eNOS (Tyr-657), and nNOS (Tyr-1326)/iNOS (Tyr-1055). The antibodies were detected using appropriate secondary antibody conjugated to DyLight® 594.



Phospho-eNOS (Tyr-657) synthetic peptide (coupled to carrier protein) corresponding to amino acids surrounding tyrosine 657 in human eNOS. This sequence is conserved in mouse (Tyr-656) and rat (Tyr-656) eNOS, and is identical to the conserved site in nNOS (Tyr-895). The site is also well conserved in iNOS (Tyr-631).

*For more information, see UniProt Accession P29474
Rabbit polyclonal, affinity-purified antibody is supplied in 100µl phosphate-buffered saline, 50% glycerol, 1 mg/ml BSA, and 0.05% sodium azide. Store at –20°C. Stable for 1 year.

The products are are safely shipped at ambient temperature for both domestic and international shipments. Each product is guaranteed to match the specifications as indicated on the corresponding technical data sheet. Please store at -20C upon arrival for long term storage.

The antibody detects a 140 kDa* band on SDS-PAGE immunoblots of human umbilical vein endothelial cells treated with pervanadate, and this reactivity is not observed after akaline phosphatase treatment.

*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blot mobilities of known proteins with similar MW.

This kit contains:

KIT SUMMARY