Anti-E Cadherin antibody [M168] - C-terminal

• WB

Supplier Data

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

Blocked and probed in the presence of 5% milk, and the image was captured using chemiluminescence and film.

Lane 1:

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (ab76055) at 1/1000 dilution

Lane 2:

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (ab76055) at 1/2000 dilution

Lane 3:

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (ab76055) at 1/4000 dilution

All lanes:

A431 (Human epidermoid carcinoma cell line) denatured whole cell lysate at 10 µg

Predicted band size: 97 kDa

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Exposure time: 2min

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

Immunohistochemistry staining of E Cadherin in human MMCR (Muller's muscle conjunctival resection) tissue.

Xylene and rehydration with serial ethanol dilutions were used for deparaffinization. Slides were washed twice for 5 minutes in 0.25% Triton X-100 for permeabilization and blocked for 2 hours at room temperature with 2% BSA and 10% normal donkey serum in PBS. Slides were incubated overnight at 4°C with ab76055. The next day, the slides were washed twice for 5 minutes in PBS and incubated for 1–2 hours with respective secondary antibodies diluted in blocking solution (1 : 200–800). Vecta shield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was placed over the slides and covered with a glass coverslip. Slides were analyzed using the Zeiss LSM 710 Confocal Microscope.

Shah et al PLoS One. 2016 Jan 29;11(1):e0148018. doi: 10.1371/journal.pone.0148018. eCollection 2016. Fig 2. Reproduced under the Creative Commons license https://creativecommons.org/publicdomain/zero/1.0/

• ICC

Lab

Immunocytochemistry - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

ICC/IF image of ab76055 stained A431 (Human epidermoid carcinoma cell line) cells.

The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76055, 1/100 dilution) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight^® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 μM.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with ab76055 (red line).

The cells were fixed with 4% paraformaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (ab76055, 1/100 dilution) for 30 minutes at 22°C. The secondary antibody used was a DyLight^® 488 goat anti-mouse IgG (H+L) ab96879 at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 μg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

This antibody gave a positive signal in A431 cells fixed with 80% methanol (5 minutes)/permeabilized with 0.1% PBS-Tween for 20 minutes used under the same conditions.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

IHC image of E Cadherin staining in human colon 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 pH 6, for 20 minutes. The section was then incubated with ab76055, 5 μg/ml, for 15 minutes 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.

• WB

CiteAb

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

Western Blotting using Anti-E Cadherin antibody [M168] - C-terminal, ab76055. Publication image from Redon, C. E. et al., 2016, Nat Commun, 26876487. Legend direct from paper.

Downregulation of the transcription factors Slug and ZEB1 reverses the EMT programme induced by H2A.X deficiency.(a) Control cells (shCTRL) and cells silenced for H2A.X (shH2A.X) were transfected for 3 days with control siRNA (siC) or with a pool of siSLUG and siZEB1 (siS/Z). Immunoblot analysis of EMT markers was performed using tubulin as loading control. (b) Staining of E-cadherin and β-catenin by immunofluorescence in HCT116 control cells (shCTRL) and H2A.X-deficient cells (shH2A.X) transfected for 3 days with control siRNA (siC) or a pool of siRNAs against SLUG and ZEB1 (siS/Z). Nuclei were counterstained with propidium iodide (red); scale bars, 20 µm. (c) Top panel, analysis of cell phenotypic changes through the staining of F-actin (phalloidin) by immunofluorescence using conditions described in b; scale bars, 20 µm. Bottom panel, photomicrographs of cells; scale bar, 100 µm.(d) Immunoblot analysis of EMT markers in HCT116 parental cells (WT) and H2A.X knockout cells (KO) transfected for 3 days with siRNA control (siC) or with a pool of siSLUG and siZEB1 (siS/Z) with tubulin-loading controls. H2A.X knockout cells were generated using CRISPR/Cas9 system for precise deletion of the H2A.X gene. cl.5, clone #5; and cl.6, clone #6. (e) Immunofluorescence assays showing E-cadherin and β-catenin staining. Cells were treated as in d. Nuclei were counterstained in red with propidium iodide; scale bars, 20 µm.

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• WB

CiteAb

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

Western Blotting using Anti-E Cadherin antibody [M168] - C-terminal, ab76055. Publication image from Redon, C. E. et al., 2016, Nat Commun, 26876487. Legend direct from paper.

H2A.X re-expression inhibits EMT and promotes metastatic colonization in the lung.(a) Immunoblot analysis of EMT markers in HCT116 parental cells (WT), H2A.X knockout cells (KO) and H2A.X knockout cells in which H2A.X expression was restored (KO+H2A.X), utilizing actin as a loading control. (b) HCT116 parental cells (WT), H2A.X knockout cells (KO) and revertant cells (KO+H2A.X) were analysed for transcripts levels of EMT markers by real-time PCR. Expression values for E-cadherin (CDH1), ZEB1, Slug, Vimentin (VIM), integrin beta 4 (ITGB4) and versican (VCAN) were normalized to 18S RNA (gene/18S ratio). Error bars represent the s.e. (n=3). Statistical significance was determined by a two-tailed, unpaired Student's t-test. The experiments were repeated three times. (c). Immunostaining of the epithelial marker E-cadherin. Nuclei were counterstained in red with propidium iodide; scale bars, 20 µm. Photomicrographs of cells are shown (top panel); scale bar, 100 µm. (d) Tail vein injections of HCT116 parental cells (WT) and H2A.X knockout cells (KO) showed similar numbers of lung metastatic foci 4 weeks post injection. Ectopic expression of H2A.X (KO+H2A.X) resulted in a 2.5-fold increase in lung macroscopic metastases. (e) Representative haematoxylin- and eosin-stained lung sections from mice injected with HCT116 parental cells (WT), H2A.X knockout cells (KO) and H2A.X knockout cells in which H2A.X expression was restored (KO+H2A.X). Black arrows indicate some macroscopic lung nodules. Scale bars, 1 mm. Statistical significance was determined by a two-tailed, unpaired Student's t-test.

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• WB

CiteAb

Western blot - Anti-E Cadherin antibody [M168] - C-terminal (AB76055)

Western Blotting using Anti-E Cadherin antibody [M168] - C-terminal, ab76055. Publication image from Redon, C. E. et al., 2016, Nat Commun, 26876487. Legend direct from paper.

Downregulation of the transcription factors Slug and ZEB1 reverses the EMT programme induced by H2A.X deficiency.(a) Control cells (shCTRL) and cells silenced for H2A.X (shH2A.X) were transfected for 3 days with control siRNA (siC) or with a pool of siSLUG and siZEB1 (siS/Z). Immunoblot analysis of EMT markers was performed using tubulin as loading control. (b) Staining of E-cadherin and β-catenin by immunofluorescence in HCT116 control cells (shCTRL) and H2A.X-deficient cells (shH2A.X) transfected for 3 days with control siRNA (siC) or a pool of siRNAs against SLUG and ZEB1 (siS/Z). Nuclei were counterstained with propidium iodide (red); scale bars, 20 µm. (c) Top panel, analysis of cell phenotypic changes through the staining of F-actin (phalloidin) by immunofluorescence using conditions described in b; scale bars, 20 µm. Bottom panel, photomicrographs of cells; scale bar, 100 µm.(d) Immunoblot analysis of EMT markers in HCT116 parental cells (WT) and H2A.X knockout cells (KO) transfected for 3 days with siRNA control (siC) or with a pool of siSLUG and siZEB1 (siS/Z) with tubulin-loading controls. H2A.X knockout cells were generated using CRISPR/Cas9 system for precise deletion of the H2A.X gene. cl.5, clone #5; and cl.6, clone #6. (e) Immunofluorescence assays showing E-cadherin and β-catenin staining. Cells were treated as in d. Nuclei were counterstained in red with propidium iodide; scale bars, 20 µm.

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