Anti-YAP1 antibody [EP1674Y] (ab52771) at 1/5000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 65 kDa

Blocking/Diluting buffer: 5% NFDM/TBST

Immunocytochemistry/Immunofluorescence analysis of MCF-7 (Human breast adenocarcinoma cell line) cells labeling YAP1 with purified ab52771 at 1/500.

Cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077).

Nuclei counterstained with DAPI (blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue sections labeling YAP1 with purified ab52771 at 1:50 dilution (1.78 µg/ml).

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody.

PBS instead of the primary antibody was used as the negative control.

ab52771 (purified) at 1:20 dilution (0.5 µg) immunoprecipitating YAP1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1: HeLa whole cell lysate 10 μg input.
Lane 2: ab52771 in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab52771 in HeLa whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Blocking/Diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling YAP1 with purified ab52771 at 1:20 dilution (10 µg/ml) (red).

Cells were fixed with 4% paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunofluorescence revealed that YAP1 expression was significantly lower in iVSD hearts compared to control hearts.

YAP1 (green), and DAPI (blue) staining are shown; Scale bar = 25 μm.

Cryosections of human heart tissue were blocked using 10% FBS for 30 min, and then incubated with anti YAP1 (ab52771, 1:200) at room temperature for 2 h. The slides were then incubated with the following secondary antibody, Alexa Fluor^® 488-conjugated anti-rabbit (Abcam, ab150073; 1:1,000 dilution). Cell nuclei were stained with DAPI.

Anti-YAP1 antibody [EP1674Y] (ab52771) at 1/50000 dilution (purified) + Human thyroid cancer lysates at 15 µg

Secondary
Rabbit monoclonal [EP1674Y] to YAP1 (ab52771) at 1/2000 dilution (Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG)

Predicted band size: 65 kDa

Blocking/Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of YAP1 in paraffin embedded human breast carcinoma tissue using unpurified ab52771 at a 1/100 dilution.

Anti-YAP1 antibody [EP1674Y] (ab52771) at 1/50000 dilution (unpurified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 65 kDa
Observed band size: 65 kDa

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with  unpurified ab52771 (red line).

The cells were fixed with 80% methanol (5 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 (ab52771, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C.

Isotype control antibody (black line) was rabbit IgG (monoclonal) (1 μg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

All lanes are unpurified ab52771 at a 1/1000 dilution plus whole cell lysate prepared from U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells, treated with DMSO or 1 µM and 10 µM LY294002, 50 µg positive control loaded.

Primary antibody was incubated for 16 hours at 4°C.

Blocking step was performed using 5% milk for 1 hour at 23°C.

Secondary used was a goat polyclonal conjugated to HRP, at a 1/10,000 dilution.

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