Figure 1: eNOS target protein structure.

eNOS Target Introduction

Protein Function

• NOS proteins are divided into three subtypes: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).
• eNOS is involved in the relaxation of vascular smooth muscle and the production of nitric oxide (NO) through the cGMP-mediated signaling pathway. NO mediates coronary vessel formation induced by vascular endothelial growth factor (VEGF) and promotes blood clotting by activating platelets.

Protein Expression

• Tissue-specific expression: eNOS is expressed in platelets, placenta, liver, and kidneys.

Protein Localization

• Located in the cell membrane, cytoskeleton, Golgi apparatus, etc.
• Particularly associated with the actin cytoskeleton during the G2 phase of the cell cycle and interacts with NOSIP, leading to decreased enzyme activity.

Figure 2: eNOS ICC experimental result image, using the Anti-eNOS antibody [EPR23750-3] (ab252439). Negative control: HeLa.

Isoforms & Post-translational modifications

• Human (P29474): There are 3 isoforms, isoform 1: 133 kDa (predicted); isoforms eNOS13C and eNOS13B: 68 kDa (predicted), with eNOS13C lacking eNOS activity.
• Mouse (P70313): 132 kDa (predicted)
• Rat (Q62600): 133 kDa (predicted)
• Phosphorylation modifications on multiple serine and threonine residues.
• Myristoylation, palmitoylation modifications.

WB experiment tips

Precautions

• eNOS protein is prone to degradation when stored in a lysis buffer. To avoid protein degradation and weakening of the detection signal, please use freshly prepared whole-cell lysate and immediately use it for WB detection.
• eNOS protein is predicted to be 133 kDa in size and a splice variant of approximately 60 kDa. Multiple bands may be observed during WB detection.
• Some cell lines may show increased detection of eNOS protein after drug stimulation (e.g. 10 µg/mL LPS stimulation of HepG2 cells for 2 hours).
• Some cell lines may show increased eNOS (phospho S1177) protein detection after drug stimulation (e.g., HUVEC cells stimulated with pervanadate or VEGF, or THP1 cells stimulated with PMA).
• When detecting phosphorylated eNOS protein, a phosphatase treatment group can be set up to determine the specificity of the phosphorylation signal.

Positive control

• eNOS: HUVEC whole cell lysate, EA.hy926 whole cell lysate. Please choose the most appropriate positive control according to the product manual.
• eNOS (phospho S1177): HUVEC (stimulated with VEGF or pervanadate) whole cell lysate, THP1 (stimulated with PMA) whole cell lysate. Please choose the most appropriate stimulating drug according to the product manual.

Negative control (no expression or weak expression)

• eNOS: HeLa whole cell lysate.

Example of results

Figure 3: WB-Anti-eNOS antibody [3/eNOS/NOS Type III] (ab282109).

Lane 1: 20 µg of HUVEC whole cell lysate
Lane 2: 20 µg of Hela whole cell lysate
Lane 3: 20 µg of EA.hy926 whole cell lysate

Primary antibody: Anti-eNOS antibody [3/eNOS/NOS Type III] (ab282109), diluted at a concentration of 1/2000.
Secondary antibody: Peroxidase-conjugated goat anti-mouse IgG (H+L), diluted at a concentration of 1/10000.
Band size detected: 140 kDa.

Figure 4: WB-Anti-eNOS (phospho S1177) antibody [EPR20991] (ab215717).

Lane 1: 10 µg of HUVEC whole cell lysate.
Lane 2: 10 µg of HUVEC whole cell lysate treated with 100 uM pervanadate for 10 minutes.

Primary antibody: eNOS (phospho S1177) antibody [EPR20991] (ab215717), diluted at 1/1000.
Secondary antibody: HRP-conjugated goat anti-rabbit IgG (H+L) (ab97051), diluted at 1/100000.
Predicted band size: 133 kDa.
Detected band size: 140 kDa, the 60 kDa band may be a splice variant of eNOS.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. For phosphorylated modified proteins, add a complex phosphatase inhibitor to prevent dephosphorylation during extraction.
3. Keep the sample on ice throughout the sample preparation process.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
5. We recommend using a positive control.

Electrophoresis:

1. For target proteins with larger molecular weight (such as molecular weight > 100 kDa), use an 8% separation gel for electrophoresis.
2. Load at least 20 μg total protein for electrophoresis.

Transfer:

1. For target proteins with larger molecular weight, it is recommended to add SDS to a final concentration of 0.1% in the transfer buffer.
2. For target proteins with larger molecular weight, we recommend using a 0.45 μm PVDF membrane.
3. For target proteins with larger molecular weight, it is recommended to use 10% methanol or lower concentration in the transfer buffer.
4. It is recommended to use Ponceau S staining after transfer to confirm the success of the transfer.

References

1. Chien-Hsing Lee, Yin-Win Wei, etc. CDK5 phosphorylates eNOS at Ser-113 and regulates NO production. J Cell Biochem. 2010 May;110(1):112-7. doi: 10.1002/jcb.22515.
2. E P Garvey, J V Tuttle, etc. Purification and characterization of the constitutive nitric oxide synthase from human placenta. Arch Biochem Biophys. 1994 Jun;311(2):235-41. doi: 10.1006/abbi.1994.1232.
3. Monique David-Dufilho, Elisabeth Millanvoye-Van Brussel, etc. Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II. J Biol Chem. 2005 Oct 28;280(43):35999-6006. doi: 10.1074/jbc.M506374200. Epub 2005 Aug 26.
4. J Dedio, P König, P Wohlfart, etc. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J. 2001 Jan;15(1): 79-89.doi: 10.1096/fj.00-0078com.
5. Stefania Bulotta, Maria Vincenza Ierardi, Jessica Maiuolo, etc. Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells. Biochem Biophys Res Commun. 2009 Sep 4;386(4):744-9. doi: 10.1016/j.bbrc.2009.06.118. Epub 2009 Jun 25.
6. Yi-Chiung Hsu 1, Hsin-Chen Lee, Yueh-Hsin Ping, etc. Mitochondria are an essential mediator of nitric oxide/cyclic guanosine 3',5'-monophosphate blocking of glucose depletion induced cytotoxicity in human HepG2 cells. Mol Cancer Res. 2007 Sep;5(9):923-32. doi: 10.1158/1541-7786.MCR-07-0026.
7. María M Arriero, Juan A Rodríguez-Feo, Ángel Celdrán, etc. Expression of endothelial nitric oxide synthase in human peritoneal tissue: regulation by Escherichia coli lipopolysaccharide. J Am Soc Nephrol. 2000 Oct;11(10):1848-1856. doi: 10.1681/ASN.V11101848.
8. Stuart A Ritchie, Christine F Kohlhaas, Alasdair R Boyd, etc. Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis. Biochem J. 2010 Jan 27;426(1):85-90. doi: 10.1042/BJ20091580.
9. M Tesauro, W C Thompson, J Moss. Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells. Cell Death Differ. 2006 Apr;13(4):597-606. doi: 10.1038/sj.cdd.4401770.