YAP - Mediator of the Hippo Signaling Pathway

YAP (Yes-associated protein, YAP65) is a transcriptional co-activator that functions as the primary effector of the Hippo tumor suppressor pathway. Hippo signaling via YAP regulates tissue growth and organ size, in part by modulating the proliferative activity of stem and progenitor cells. YAP is a potent oncogene whose aberrant regulation can lead to tumorigenesis, and elevated YAP expression has been reported in many human tumor types. Research studies also suggest that YAP plays an important role in cell fate determination. In addition to its reported ability to promote epithelial-mesenchymal transition (1), YAP was shown to promote dedifferentiation of mature hepatocytes into hepatic progenitor cells, suggesting an important role for YAP and Hippo signaling in tissue regeneration (2).

Lamar JM, Stern P, Liu H, Schindler JW, Jiang ZG, Hynes RO (2012) The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proc. Natl. Acad. Sci. U.S.A. 109(37), E2441–50.
Yimlamai D, Christodoulou C, Galli GG, Yanger K, Pepe-Mooney B, Gurung B, Shrestha K, Cahan P, Stanger BZ, Camargo FD (2014) Hippo pathway activity influences liver cell fate. Cell 157(6), 1324–38.

Antibody Performance Comparisons

We compared our YAP (D8H1X) XP^® Rabbit mAb #14074 with 3 YAP antibodies from other companies and found that it performed as well or better in every application tested. See competitor comparison data below for individual applications.

Western Blotting

Western blot analysis of extracts from YAP positive cells (A-204, A549, MCF7) and YAP negative cells (RL-7) using our YAP (D8H1X) XP^® Rabbit mAb #14074, and three other antibodies at the recommended dilutions.

YAP (D8H1X) XP^® Rabbit mAb #14074 results in a band at the appropriate molecular weight for YAP in YAP positive cells and no bands in YAP negative cells, indicating that it is sensitive and specific for YAP.
Antibody 1 gives a strong signal for YAP, but strongly cross-reacts with a 50 kDa protein suggesting that this antibody is not specific for YAP.
Antibody 2 produces a weak signal for YAP at the dilution recommended by the supplier.
Antibody 3 gives bands at multiple molecular weights for both YAP positive and YAP negative cell lines, demonstrating that this antibody is not specific for YAP.

Summary: YAP (D8H1X) XP^® Rabbit mAb #14074 demonstrates higher specificity and sensitivity than the other antibodies.

YAP Antibody Performance Comparison: Flow Cytometry

Of the antibodies we tested by western blot, only YAP (D8H1X) XP^® Rabbit mAb #14074 and Antibody 2 rabbit monoclonal antibody are recommended for flow cytometry. We analyzed YAP positive A-204 cells (green) and YAP negative RL-7 cells (blue) using YAP (D8H1X) XP^® Rabbit mAb #14074 and Antibody 2 at the recommended dilutions.

YAP (D8H1X) XP^® Rabbit mAb #14074 results in a good separation of fluorescence intensity peaks between the negative and positive cells, demonstrating a high specificity for YAP.
At the same instrumental settings, Antibody 2 results in high signal for YAP-positive cells but also higher background signal in YAP-negative cells, suggestive of non-specifc reactivity.

Summary: YAP (D8H1X) XP^® Rabbit mAb #14074 demonstrates higher specificity than Antibody 2.

YAP Antibody Performance Comparison: IHC

Cell Pellets

IHC analysis of paraffin-embedded YAP positive (PANC-1) and YAP negative (RL-7) cell pellets using YAP (D8H1X) XP^® Rabbit mAb #14074 and antibodies from two other companies.

CST YAP (D8H1X) XP^® Rabbit mAb #14074 gives strong cytoplasmic and nuclear staining in YAP positive cells and no staining in YAP negative cells, demonstrating that this antibody is specific for YAP.
Antibody 1 demonstrates appropriate staining in YAP positive cells and no staining in YAP negative cells. More antibody is used per assay suggesting that #14074 is more sensitive than Antibody 1.
Antibody 3 stains YAP negative cells at 1:4000 demonstrating that it is not specific for YAP. At 1:8000 very little staining is seen in the YAP negative cells, but the signal is also reduced in YAP positive cells. #14074 is more specific and sensitive than Antibody 3.

Breast Carcinoma

IHC analysis of paraffin-embedded breast carcinoma using YAP (D8H1X) XP^® Rabbit mAb #14074 at the recommended dilution and a mouse monoclonal antibody from another company at the dilution that we determined to be optimal based on the cell pellet analysis.

CST YAP (D8H1X) XP^® Rabbit mAb #14074 results in strong staining in paraffin-embedded breast carcinoma.
Antibody 1 gives very weak staining at 1:10 dilution.

Summary: Using cell pellets and paraffin-embedded breast carcinoma we demonstrate that YAP (D8H1X) XP^® Rabbit mAb #14074 yields superior sensitivity and specificity in IHC assays than the antibodies from other companies.

YAP Antibody Performance Comparison: IF

IF-IC analysis of MCF10A and C2C12 cells using YAP (D8H1X) XP^® Rabbit mAb #14074 and antibodies from two other companies.

MCF10A Human Mammary Epithelial Cells

Antibody 1 appears to perform equally as well as #14074, but requires much more antibody to achieve similar intensity.
Antibody 3 gives very weak staining at the dilution recommended by the supplier.

C2C12 Mouse Myoblast Cells

CST YAP (D8H1X) XP^® Rabbit mAb #14074 and Antibody 1 result in appropriate staining for YAP with no staining in the nucleolus.
In addition to weak nuclear staining, Antibody 3 results in strong staining of the nucleolus, demonstrating a lack of specificity of this antibody for YAP in rodent cell models.

Summary: YAP (D8H1X) XP^® is specific in human and mouse cells while Antibody 3 demonstrates non-specific staining in rodent cells. YAP (D8H1X) XP^® Rabbit mAb #14074 has higher specific activity than Antibody 1. In addition, Antibody 1 cross reacts strongly with a 50 kDa protein by western blot and it is possible that it also cross reacts with this protein by immunofluorescence.