Anti-Ezrin antibody [3C12]

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Ezrin antibody [3C12] (AB4069)

IHC image of ab4069 staining in 10% formaldehyde fixed frozen tissue section of human placenta.

Non-specific protein-protein interactions were blocked using TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1 hour at room temperature. The section was then incubated with ab4069 (1μg/ml dilution) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. The section was then incubated with ab150119 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647)) and DAPI for 1 hour at room temperature.

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is derived directly from bound ab4069.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ezrin antibody [3C12] (AB4069)

Overlay histogram showing SH-SY5Y cells stained with ab4069 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab4069, 1μg/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 positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [3C12] (AB4069)

IHC image of Ezrin staining in a section of formalin-fixed paraffin-embedded normal human lung 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 20mins. The section was then incubated with ab4069, 0.025ug/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [3C12] (AB4069)

IHC image of Ezrin staining in mouse lung formalin fixed paraffin embedded tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab4069, 1μg/ml overnight at +4°C. An HRP-conjugated secondary (ab97040, 1/1000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

• WB

Lab

Western blot - Anti-Ezrin antibody [3C12] (AB4069)

Lanes 1 - 5 : Merged signal (red and green). Green - ab4069 observed at 81 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab4069 was shown to specifically react with Ezrin in wild-type HAP1 cells as signal was lost in EZR (Ezrin) knockout cells. Wild-type and EZR (Ezrin) knockout samples were subjected to SDS-PAGE. ab4069 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Ezrin antibody [3C12] (ab4069) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

EZR (Ezrin) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MEF whole cell lysate at 20 µg

Lane 5:

NIH3T3 whole cell lysate at 20 µg

Predicted band size: 69 kDa

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• WB

AbReview44010****

Western blot - Anti-Ezrin antibody [3C12] (AB4069)

All lanes:

Western blot - Anti-Ezrin antibody [3C12] (ab4069) at 1/500 dilution

All lanes:

Mouse bEnd.3 whole cell lysate at 20 µg

Secondary

All lanes:

HRP-conjugated goat anti-mouse IgG polyclonal at 1/10000 dilution

Predicted band size: 69 kDa

Observed band size: 81 kDa

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Exposure time: 10s

This image is courtesy of an anonymous Abreview

• WB

CiteAb

Western blot - Anti-Ezrin antibody [3C12] (AB4069)

Ezrin western blot using anti-Ezrin antibody [3C12] ab4069. Publication image and figure legend from Miura, S., Sato, K., et al., 2015, Nat Commun, PubMed 26563429.

ab4069 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 ab4069 please see the product overview.

Essential roles of TRPV6 in FSS-induced microvilli formation.(a) Apical localization of ezrin in the FSS-exposed BeWo cells. Cells were cultured overnight under static conditions, and then cultured with or without medium perfusion (5 μl min−1) for 1 h. Ezrin (green) localization was observed by immunofluorescence confocal microscopy. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Serial x–y focal planes (z-sections, 0.88 μm interval in ‘Static', 0.90 μm in ‘Flow') and the stacked images are shown. Scale bar, 20 μm. (b) Time-course phosphorylation of Ezrin (pThr567) and Akt (pSer473) in response to FSS. BeWo cells were cultured overnight under static conditions and exposed to FSS (5 μl min−1) for the indicated times. The cells were lysed, and protein expression level or phosphorylation was analysed by immunoblot. (c) Inhibition of Akt phosphorylation by BAPTA-AM. Gö6793 (100 nM), BAPTA-AM (10 μM) or buffer control (DMSO) was added to the perfusing medium. (d) Involvement of TRPV6 in the FSS-induced Ezrin phosphorylation. BeWo cells transfected with siRNA were seeded in the chamber area of the device and cultured overnight under static conditions before exposure to FSS for 1 h. β-Actin was used as a loading control. Note that the TRPV6 siRNA oligo (TRPV6 si-#1 or #2), as well as the siRNA pool, shows similar inhibitory effects on the FSS-induced phosphorylation of Ezrin and Akt. (e) Impaired Ezrin localization in the TRPV6 knockdown cells. Ezrin localization in the TRPV6 knockdown cells was analysed by immunofluorescence confocal microscopy. Note that TRPV6 knockdown cells exposed to FSS (5 μl min−1) for 1 h failed to show the microvillous localization pattern of Ezrin as observed in the control siRNA cells under FSS. Scale bar, 20 μm.

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