Anti-NOX2/gp91phox antibody [54.1]

• WB

CiteAb

Western blot - Anti-NOX2/gp91phox antibody [54.1] (AB80897)

NOX2/gp91phox western blot using anti-NOX2/gp91phox antibody [54.1] ab80897. Publication image and figure legend from Dingjan, I., Verboogen, D. R., et al., 2016, Sci Rep, PubMed 26907999.

ab80897 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab80897 please see the product overview.

NOX2-produced ROS cause endosomal lipid peroxidation.(a) H2O2 production by DCs cultured in absence (no LPS; blue curve) or presence of LPS (+LPS; black) measured with Amplex Red. (b) Sensor Bodipy581/591-C11 (structure) increases fluorescence at 510 nm upon peroxidation. Histograms show fluorescence intensity distributions from a typical experiment of DCs labeled with Bodipy581/591-C11 and incubation with LPS for the times indicated. (c–d) The mean fluorescence intensities (MFI) measured by flow cytometry of DCs cultured for 60 min with Bodipy581/591-C11 in presence or absence of LPS (c), or with LPS in absence (Ctrl) or presence of α-tocopherol (α-Toc) or phenylarsine oxide (PAO) (d). (e) Quantification of knock-down efficiency for gp91phox (NOX2KD) by Western blot (NT : non-targeting siRNA; GAPDH : loading control). (f) Similar to c, but now for NOX2KD DCs. (g) Similar to d, but now for mouse BMDCs. (h) Confocal microscope images of DCs stained for gp91phox (green) after treatment with OVA conjugated with Alexa fluor 647 (OVA-AF647; red). (i) Detection of peroxidation-modified proteins on endosomes. DCs were incubated with OVA-AF647 and linoleamide alkyne (LAA). After incubation, cells were fixed and stained with Alexa fluor 488 azide (LAA-AF488). The fluorescence threshold was set on the OVA-AF647 channel and the green fluorescence was quantified. BF : bright field. (j) Difference between green fluorescence of the whole cell and the fluorescence thresholded on OVA-AF647. (k) LAA assay for DCs cultured in the absence or presence of LPS. LAA-AF488 signal was quantified for at least 10 cells. (l) Same as panel (k), but now for LPS-treated DCs in the absence or presence of α-Toc. Results show individual results for at least 3 donors/mice. Results from the same donor/mouse are connected by a black line. Scale bars, 10 μm.

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOX2/gp91phox antibody [54.1] (AB80897)

IHC image of ab80897 staining in normal human skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 ab80897, 5µg/ml, 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NOX2/gp91phox antibody [54.1] (AB80897)

ICC/IF image of ab80897 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab80897, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• WB

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Western blot - Anti-NOX2/gp91phox antibody [54.1] (AB80897)

All lanes:

Western blot - Anti-NOX2/gp91phox antibody [54.1] (ab80897) at 1 µg/mL

Lane 1:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg

Lane 2:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

Human lymph node tissue lysate - total protein (<a href='/en-us/products/unavailable/human-lymph-node-tissue-lysate-total-protein-ab29871'>ab29871</a>) at 10 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 65 kDa

Observed band size: 29 kDa,35 kDa,65 kDa

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Exposure time: 12min