Catalog # NK6430

eNOS Phospho-Regulation Antibody Kit

Antibody Sampler Kit



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 O2) 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.

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

Western blot analysis of human umbilical vein endothelial cells treated with calyculin A (100 nM) for 30 min. (lanes 1, 3, 5, & 7) then the blots were treated with lambda phosphatase (lanes 2, 4, 6, & 8). The blots were probed with anti-endothelial nitric oxide synthase (eNOS) (C-terminal region) (lanes 1 & 2), anti-eNOS (Ser-632) (lanes 3 & 4), anti-eNOS (Ser-1177) (lanes 5 & 6), or anti-eNOS (a.a. 1172-1181) (lanes 7 & 8).

Immunocytochemical labeling of endothelial nitric oxide synthase (eNOS) in paraformaldehyde-fixed and NP-40-permeabilized human umbilical vein endothelial cells. The cells were labeled with mouse monoclonal eNOS (NM2211) and rabbit polyclonal eNOS (NP2281) antibodies, then the antibodies were detected using appropriate secondary antibodies conjugated to Cy3.

Rabbit polyclonal and mouse monoclonal antibodies are supplied in 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.

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

Product References:

NP4051 Pratumvinit, B. et al. (2013) Biomed Res Int. 2013:359412. WB: mouse cardiac endothelial cells

This kit contains:

eNOS (C-terminal region) Mouse mAb50 μlWB, E, ICC, IHCHu140
eNOS (a.a. 1172-1181) Rabbit pAb50 μlWB, EHu, Rt, Ms140
eNOS (Ser-632), phospho-specific Mouse mAb50 μlWB, EHu, Rt, Ms140
eNOS (Tyr-657/nNOS (Tyr-895), phospho-specific Rabbit pAb50 μlWB, E, ICCHu, Rt, Ms140
eNOS (Ser-1177), phospho-specific Rabbit pAb50 μlWB, EHu, Rt, Ms140

The endothelial nitric oxide synthase (eNOS) phospho-regulation antibody sampler kit can be used to examine phosphorylation of eNOS at Ser-632, Tyr-657, and Ser-1177. The kit also includes a mouse monoclonal and rabbit polyclonal antibody to monitor total expression levels of eNOS.