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56612
Hippo Pathway: Upstream Signaling Antibody Sampler Kit

Hippo Pathway: Upstream Signaling Antibody Sampler Kit #56612

Western Blotting Image 1

Western blot analysis of extracts from various cell lines using YAP (D8H1X) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, RL-7 cells are negative for YAP protein expression.

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Chromatin IP-seq Image 2

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCI-H2052 cells and YAP (D8H1X) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 5ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across CTGF, a known target gene of YAP (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 3

Western blot analysis of extracts from various cells using TAZ (D3I6D) Rabbit mAb (upper) and GAPDH (D16H11) XP® #5174 (lower). As expected, Raji cells are negative for TAZ.

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Chromatin IP-seq Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCI-H2052 cells and TAZ (D3I6D) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across SMYD3, a known target gene of TAZ (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using Ajuba (D4D8P) Rabbit mAb.

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using PTPN14 (D5T6Y) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 7

Western blot analysis of extracts from PC-3 cells, and both wild type and Nf2 (-/-) mouse embryonic fibroblasts (MEFs) using Merlin (D3S3W) Rabbit mAb (upper), Ezrin/Radixin/Moesin Antibody #3142 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower). (MEF wt and MEF Nf2 (-/-) cells were kindly provided by Dr. Andrea McClatchey and Dr. Andrew Gladden, MGH Cancer Center and Harvard Medical School, Charlestown MA).

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using MST1 (D8B9Q) Rabbit mAb.

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Western Blotting Image 9

Western blot analysis of extracts from various cell lines using FRMD6 (D8X3R) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 10

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IP Image 11

Immunoprecipitation of YAP protein from A-204 cell extracts using Rabbit (DA1E) mAb XP® Isotype Control #3900 (lane 2) or YAP (D8H1X) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using YAP (D8H1X) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.

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Chromatin IP Image 12

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCI-2052 cells and either YAP (D8H1X) XP® Rabbit mAb, or Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CTGF Promoter Primers #14927, human SMYD3 intron 2 primers, and SimpleChIP® Human CTGF Upstream Primers #14928. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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Chromatin IP Image 13

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCI-H2052 cells and either 10 μl of TAZ (D3I6D) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CTGF Promoter Primers #14927, human SMYD3 intron 2 primers, and SimpleChIP® Human CTGF Upstream Primers #14928. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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IP Image 14

Immunoprecipitation of Ajuba protein from PANC-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb XP® Isotype Control #3900, and lane 3 is Ajuba (D4D8P) Rabbit mAb. Western blot analysis was performed using Ajuba (D4D8P) Rabbit mAb.

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IP Image 15

Immunoprecipitation of merlin from mouse embryonic fibroblasts (MEF) cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Merlin (D3S3W) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Merlin (D3S3W) Rabbit mAb.

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Western Blotting Image 16

Western blot analysis of extracts from HeLa cells, serum-starved overnight and then vehicle treated (-) or treated with Staurosporine #9953 (1 μM, 4 hr; +), using MST1 (D8B9Q) Rabbit mAb. Staurosporine induces caspase-mediated cleavage of full-length MST1, yielding a 37 kDa amino-terminal fragment detected by this antibody, and an undetected 18 kDa carboxy-terminal fragment.

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human breast adenocarcinoma using YAP (D8H1X) XP® Rabbit mAb.

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IF-IC Image 18

Confocal immunofluorescent analysis of PC-3 cells using Merlin (D3S3W) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IP Image 19

Immunoprecipitation of MST1 from Ramos cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or MST1 (D8B9Q) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using MST1 (D8B9Q) Rabbit mAb.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human breast adenocarcinoma using YAP (D8H1X) XP® Rabbit mAb.

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using YAP (D8H1X) XP® Rabbit mAb.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded PANC-1 (left) and RL-7 (right) cell pellets using YAP (D8H1X) XP® Rabbit mAb.

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded human benign prostatic hyperplasia using YAP (D8H1X) XP® Rabbit mAb.

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Flow Cytometry Image 24

Flow cytometric analysis of RL-7 cells (blue) and A-204 cells (green) using YAP (D8H1X) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. 

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IF-IC Image 25

Confocal immunofluorescent analysis of low confluence MCF 10A cells (left), high confluence MCF 10A (center), and YAP negative RL-7 cells (right) using YAP (D8H1X) XP® Rabbit mAb (green). Blue pseudocolor in lower images = DRAQ5® #4084 (fluorescent DNA dye). Increased nuclear localization of YAP protein is seen in low confluence (proliferating) cells.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
YAP (D8H1X) XP® Rabbit mAb 14074 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Hm Mk 65-75 Rabbit IgG
TAZ (D3I6D) Rabbit mAb 70148 20 µl
  • WB
  • IP
  • ChIP
H M 50 Rabbit IgG
Ajuba (D4D8P) Rabbit mAb 34648 20 µl
  • WB
  • IP
H Mk 55 Rabbit IgG
PTPN14 (D5T6Y) Rabbit mAb 13808 20 µl
  • WB
H M R 160 Rabbit IgG
Merlin (D3S3W) Rabbit mAb 12888 20 µl
  • WB
  • IP
  • IF
H M R Hm Mk 70 Rabbit IgG
MST1 (D8B9Q) Rabbit mAb 14946 20 µl
  • WB
  • IP
H M R 60 Rabbit IgG
FRMD6 (D8X3R) Rabbit mAb 14688 20 µl
  • WB
  • IP
H M R 78 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Hippo Pathway Proteins Antibody Sampler Kit provides an economical means of detecting proteins that have been identified as upstream regulators of the Hippo Signaling Pathway. The kit provides enough antibody to perform two western blot experiments with each primary antibody.

Each antibody in the Hippo Pathway Proteins Antibody Sampler Kit detects endogenous levels of its target protein.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ala200 of mouse TAZ, Phe608 of human FRMD6, Leu737 of human PTPN14, Gln470 of human merlin, Arg77 of human Ajuba, Pro9 of human MST1, and recombinant protein corresponding to the carboxy terminus of human YAP protein. The sequence region surrounding the YAP epitope is 100% conserved among all known isoforms of YAP.

The Hippo Signaling Pathway is an evolutionarily conserved signaling cascade that regulates cell, tissue and organ growth in response to diverse environmental cues, including cell density, mechanical stress, growth factors and cytokines, and metabolic signaling (1,2). In mammals, the core components of this pathway are YAP and TAZ, transcriptional co-activators that regulate the expression of target genes that control cell proliferation and survival (3). The transcriptional control of YAP/TAZ target genes is regulated through a complex kinase cascade involving mammalian sterile-20-like kinases (MST1/2), LATS1/2 kinases and key adaptor proteins. Phosphorylation of YAP and TAZ by LATS kinases results in cytoplasmic sequestration of YAP and TAZ, inhibiting transcriptional activation of their target genes. A number of proteins have been identified that function upstream of MST1/2 and LATS1/2 to influence LATS-mediated phosphorylation of YAP/TAZ. These include the tumor suppressor protein Merlin (4), the FERM-domain containing protein 6 (FRMD6/Willin) (5), the LIM-domain containing adaptor protein Ajuba (6,7), and the protein tyrosine phosphatase PTPN14 (8).

  1. Meng, Z. et al. (2016) Genes Dev 30, 1-17.
  2. Watt, K.I. et al. (2017) Front Physiol 8, 942.
  3. Fu, V. et al. (2017) Curr Opin Cell Biol 49, 99-107.
  4. Su, T. et al. (2017) Dev Cell 40, 478-490.
  5. Das Thakur, M. et al. (2010) Curr Biol 20, 657-62.
  6. Liu, X. et al. (2013) Oncogene 32, 1266-73.
  7. Reddy, B.V. and Irvine, K.D. (2013) Dev Cell 24, 459-71.
  8. Angus, L. et al. (2012) Oncogene 31, 238-50.
Entrez-Gene Id
84962 , 122786 , 4771 , 6789 , 5784 , 25937 , 10413
Swiss-Prot Acc.
Q96IF1 , Q96NE9 , P35240 , Q13043 , Q15678 , Q9GZV5 , P46937
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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