All lanes : Anti-active YAP1 antibody [EPR19812] (ab205270) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : active YAP1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 54 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab205270 observed at 54 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab205270 was shown to recognize active AP1 in wild-type HAP1 cells as signal was lost at the expected MW in active YAP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and active YAP1 knockout samples were subjected to SDS-PAGE. Ab205270 and ab9484 (Mouse 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-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling active YAP1 with ab205270 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Mainly nuclear staining on human breast is observed [PMID: 18617895].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Ab205270 staining active YAP1 in HUVEC (human umbilical vein endothelial cell) cells by Immunocytochemistry/Immunofluorescence (ICC/IF). The cells were fixed 4% Paraformaldehyde and permeabilized with 0.1% TritonX-100. Samples were incubated with primary antibody at 1:500 dilution. An Alexa Fluor^® 488 Goat anti-Rabbit was used as a secondary antibody at 1:1000 dilution. DAPI was used as a nuclear counter stain. Confocal image showing nuclear and cytoplasmic staining in HUVEC cells.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton/PBS permeabilized (RT, 5 mins) 293A (Human epithelial cell line from embryonic kidney transformed with sheared human adenovirus type 5 DNA) cells labeling active YAP1 with ab205270 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 594) secondary antibody at 1/1000 dilution.

The images showed weak staining on 293A cells under serum starvation overnight. After 10% FBS was added to the medium for 1h, the nuclear staining was increased.

The data was kindly provided by our collaborator Dr. Bin Zhao (Zhejiang University).

The nuclear counterstain is DAPI (blue). Counterstained with Phalloidin-technology^® Alexa Fluor 488 at 1/1000 dilution.

All lanes : Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1 : 293A cells under serum starvation overnight lysate
Lane 2 : 293A cell lysate under serum starvation overnight, then 10% FBS was added to medium for 1 hour

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Serum starvation induces active YAP1 Ser127 phosphorylation. The level of active YAP1 protein is inversely proportional to p-YAP1 Ser127 level (PMID: 22884261).

All lanes : Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1 : Human kidney lysate
Lane 2 : Human skin lysate
Lane 3 : HaCaT (Human keratinocyte cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution
Lane 3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2: 8 seconds; Lane 3: 30 seconds.

All lanes : Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1 : Mouse testis lysate
Lane 2 : Mouse skin lysate
Lane 3 : Mouse liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 10 seconds; Lane 2: 8 seconds; Lane 3: 3 minutes.

All lanes : Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1 : 293A cell lysate serum starved overnight and then 10% FBS added to the medium for 1 hour
Lane 2 : 293A cell lysate serum starved overnight
Lane 3 : 293A cell lysate serum starved overnight and then treated with Lambda phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : donkey anti-rabbit at 1/1000 dilution

Predicted band size: 54 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% BSA/TBST.

The two panels for pYAPS127 were just shorter and longer exposure.

The data was kindly provided by our collaborator Dr. Bin Zhao (Zhejiang University).

All lanes : Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1 : 293A cell lysate
Lane 2 : YAP/TAZ knockout 293A cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : donkey anti-rabbit at 1/1000 dilution

Predicted band size: 54 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% BSA/TBST.

The data was kindly provided by our collaborator Dr. Bin Zhao (Zhejiang University).

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling active YAP1 with ab205270 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on human breast cancer is observed [PMID: 24559095].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse skin tissue labeling active YAP1 with ab205270 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Mainly nuclear staining on mouse skin is observed [PMID: 21610251].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton/PBS permeabilized (RT, 5 mins) 293A (Human epithelial cell line from embryonic kidney transformed with sheared human adenovirus type 5 DNA) cells labeling active YAP1 with ab205270 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 594) secondary antibody at 1/1000 dilution.

The images showed nuclear staining on 293A cells, and background staining on YAP/TAZ knockout 293A cells.

The data was kindly provided by our collaborator Dr. Bin Zhao (Zhejiang University).

The nuclear counterstain is DAPI (blue). Counterstained with Phalloidin-technology^® Alexa Fluor 488 at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling active YAP1 with ab205270 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative control: no staining on human liver [PMID:17974916].

Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.