Western blot analysis of extracts from bovine aoric endothelial cells (BAEC), untreated or treated with lamda phosphatase, using Phospho-eNOS (Ser113) Antibody (upper) or eNOS antibody (lower).
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 BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
Phospho-eNOS (Ser113) Antibody detects endogenous levels of eNOS only when phosphorylated at serine 113.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser113 of human eNOS. Antibodies are purified by protein A and peptide affinity chromatography.
Endothelial nitric-oxide synthase (eNOS) is an important enzyme in the cardiovascular system. It catalyzes the production of nitric oxide (NO), a key regulator of blood pressure, vascular remodeling, and angiogenesis (1,2). The activity of eNOS is regulated by phosphorylation at multiple sites. The two most thoroughly studied sites are the activation site Ser1177 and the inhibitory site Thr495 (3). Several protein kinases including Akt/PKB, PKA, and AMPK activate eNOS by phosphorylating Ser1177 in response to various stimuli (4,5). In contrast, bradykinin and H2O2 activate eNOS activity by promoting both Ser1177 phosphorylation and Thr495 dephosphorylation (6,7).
eNOS is activated by VEGF, and this activation is associated with dephsophorylation of eNOS at serine 113. Cyclosporin A blocks this dephosphorylation of eNOS upon VEGF stimulation (8).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.