Anti-eNOS antibody [M221]

• WB

Lab

Western blot - Anti-eNOS antibody [M221] (AB76198)

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.

All lanes:

Western blot - Anti-eNOS antibody [M221] (ab76198) at 1/500 dilution

Lane 1:

Huvec cell lysates at 10 µg

Lane 2:

Mouse placenta lysates at 10 µg

Secondary

All lanes:

Goat anti Mouse IR680 at 1/10000 dilution

Predicted band size: 133 kDa

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• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-eNOS antibody [M221] (AB76198)

ab76198 staining eNOS in human umbilical vein endothelial cells. Cells with fixed with paraformaldehyde.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [M221] (AB76198)

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

• WB

Unknown

Western blot - Anti-eNOS antibody [M221] (AB76198)

All lanes:

Western blot - 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

Predicted band size: 133 kDa

Observed band size: 140 kDa

false

• WB

CiteAb

Western blot - Anti-eNOS antibody [M221] (AB76198)

Western Blotting using Anti-eNOS antibody [M221], ab76198. Publication image from Cashman, N. et al., 2017, Mol Neurodegener, 28830501. Legend direct from paper.

HDL suppression of Aβ-induced inflammation is independent of eNOS and S1P. a Intracellular NO production was measured by treating hCMEC/D3 with 100 µg/mL HDL in the presence of 1 µM DAF-2 for 6 h. Fluorescence was measured at 485 nm. b Phosphorylation of eNOS was measured by treating hCMEC/D3 with 100 µg/mL HDL for 15 min before immunoblotting for phosphorylated eNOS (p-eNOS) or total eNOS. Representative immunoblots are shown in (c). hCMEC/D3 were pretreated for 1 h with the eNOS inhibitor L-NAME (d-f) or the S1P1 and S1P3 inhibitor VPC23019 (g-i) followed by 100 µg/mL HDL for 2 h. Cells were then stimulated with 0.1 µM Aβ40 (d,g) Aβ42 monomers, (e,h) or 1 ng/mL of TNF-α (f,i) for 3 h before testing PBMC adherence. Graphs represent means ± SD of adhered PBMC relative to vehicle treated cells for at least 5 independent trials. *p < 0.05, **p < 0.01, ***p < 0.001 * p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle, § p < 0.05, §§ p < 0.01, §§§p < 0.001 versus Aβ or TNF-α

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