Anti-active YAP1 antibody [EPR19812]

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

Blocking/Dilution buffer : 5% BSA/TBST.

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

All lanes:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1:

293A cell lysate at 20 µg

Lane 2:

YAP/TAZ knockout 293A cell lysate at 20 µg

Secondary

All lanes:

donkey anti-rabbit at 1/1000 dilution

Predicted band size: 54 kDa

Observed band size: 75 kDa

false

Exposure time: 30s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• IHC

Lab

Immunohistochemistry - Anti-active YAP1 antibody [EPR19812] (AB205270)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling active YAP1 with ab205270 at a concentration of 0.09 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 32 mins.

ab205270 Anti-active YAP1 antibody [EPR19812] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-active YAP1 antibody [EPR19812] (AB205270)

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.

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HeLa treated with 100ng/ml Calyculin A for 30 min whole cell lysate at 15 µg

Lane 3:

HeLa treated with 100ng/ml Calyculin A for 30 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 75 kDa

true

Exposure time: 80s

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1:

Human kidney lysate at 10 µg

Lane 2:

Human skin lysate at 10 µg

Lane 3:

HaCaT (Human keratinocyte cell line) whole cell lysate at 10 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Lane 3:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 54 kDa

Observed band size: 75 kDa

false

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

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:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1:

293A cells under serum starvation overnight lysate at 20 µg

Lane 2:

293A cell lysate under serum starvation overnight, then 10% FBS was added to medium for 1 hour at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 54 kDa

Observed band size: 75 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

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 YAP1 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.

All lanes:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

YAP1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

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:

Western blot - 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 at 10 µg

Lane 2:

293A cell lysate serum starved overnight at 10 µg

Lane 3:

293A cell lysate serum starved overnight and then treated with Lambda phosphatase at 10 µg

Secondary

All lanes:

donkey anti-rabbit at 1/1000 dilution

Predicted band size: 54 kDa

Observed band size: 75 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1:

Mouse testis lysate at 10 µg

Lane 2:

Mouse skin lysate at 10 µg

Lane 3:

Mouse liver lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 52 kDa,54 kDa

Observed band size: 52 kDa,75 kDa

false

• WB

Supplier Data

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-active YAP1 antibody [EPR19812] (ab205270) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

Lane 2:

NIH/3T3 treated with 100ng/ml Calyculin A for 30 min whole cell lysate at 15 µg

Lane 3:

NIH/3T3 treated with 100ng/ml Calyculin A for 30 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 75 kDa

true

Exposure time: 20s

• WB

CiteAb

Western blot - Anti-active YAP1 antibody [EPR19812] (AB205270)

active YAP1 western blot using anti-active YAP1 antibody [EPR19812] ab205270. Publication image and figure legend from Chi, Y., Gong, Z., et al., 2020, J Transl Med, PubMed 32169080.

ab205270 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 ab205270 please see the product overview.

Silencing lncARSR alleviates lipid accumulation in HFD-fed mice and inhibits tumor formation in nude mice. HFD-fed mice were injected with lentivirus of sh-lncARSR or sh-NC. a LncARSR expression in mice detected by RT-qPCR. b Lipid accumulation in mice determined by H&E staining and Oil Red-O staining (×200). c TG content in mice. d IRS2 expression in mice detected by RT-qPCR. e Expression YAP1, IRS2 and AKT and phosphorylation of YAP1 and AKT levels detected by western blot analysis. f Expression of adipogenesis related proteins (Fasn, Scd1 and GPA) in mice detected by western blot analysis. HFD-fed mice were injected with HepG2 cells stably transfected with sh-lncARSR or sh-NC. g Tumor size and volume examined in xenograft tumor model. n = 15 *p < 0.05 against NAFLD mice injected with sh-NC lentivirus or sh-NC-transfected HepG2 cells. Differences between two groups were analyzed by unpaired t-test. Results were expressed as mean ± standard deviation

false