Recombinant fragment of mouse eNOS protein that included amino acid residues in the C terminal region.
Human umbilical vein endothelial cells untreated and treated with lambda phosphatase. Mouse placenta lysate. Huvec lysate.
IHC-P: FFPE human normal placenta tissue sections.
This antibody detects eNOS in mouse and rat but at a lower intensity than in human. If you are working in mouse or rat, we would recommend using no more than 1% milk as the blocking agent for optimal signal strength.
Storage instructionsShipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.
FunctionProduces nitric oxide (NO) which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets. Isoform eNOS13C: Lacks eNOS activity, dominant-negative form that may down-regulate eNOS activity by forming heterodimers with isoform 1.
Tissue specificityPlatelets, placenta, liver and kidney.
Involvement in diseaseVariation in NOS3 seem to be associated with susceptibility to coronary spasm.
Sequence similaritiesBelongs to the NOS family. Contains 1 FAD-binding FR-type domain. Contains 1 flavodoxin-like domain.
Post-translational modificationsPhosphorylation by AMPK at Ser-1177 in the presence of Ca(2+)-calmodulin (CaM) activates activity. In absence of Ca(2+)-calmodulin, AMPK also phosphorylates Thr-495, resulting in inhibition of activity (By similarity). Phosphorylation of Ser-114 by CDK5 reduces activity.
Cellular localizationCell membrane. Membrane, caveola. Cytoplasm, cytoskeleton. Golgi apparatus. Specifically associates with actin cytoskeleton in the G2 phase of the cell cycle and which is favored by interaction with NOSIP and results in a reduced enzymatic activity.
Western blot - Anti-eNOS antibody [M221] (ab76198)
All lanes : Anti-eNOS antibody [M221] (ab76198) at 1/500 dilution
Lane 1 : Huvec cell lysates Lane 2 : Mouse placenta lysates
Lysates/proteins at 10 µg per lane.
Secondary Goat anti Mouse IR680 at 1/10000 dilution
Predicted band size : 133 kDa
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab76198 overnight at 4°C. Antibody binding was detected using Goat anti Mouse IR680 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
IHC image of eNOS staining in human normal placenta*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab76198, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
ab76198 staining eNOS in human umbilical vein endothelial cells. Cells with fixed with paraformaldehyde.
Flow Cytometry-eNOS antibody [M221](ab76198)
Overlay histogram showing HEK293 cells stained with ab76198 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76198, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
Western blot - eNOS antibody [M221] - C-terminal (ab76198)
All lanes : Anti-eNOS antibody [M221] (ab76198) at 1/1000 dilution
Lane 1 : human umbilical vein endothelial
cells, untreated Lane 2 : human umbilical vein endothelial
cells, treated with lambda phosphatase
References for Anti-eNOS antibody [M221] (ab76198)
This product has been referenced in:
Song CL et al. MicroRNA-130a alleviates human coronary artery endothelial cell injury and inflammatory responses by targeting PTEN via activating PI3K/Akt/eNOS signaling pathway. Oncotarget7:71922-71936 (2016).
Read more (PubMed: 27713121) »
Stratmann B et al. Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates. Sci Rep6:37737 (2016).
Read more (PubMed: 27898103) »