Name: Hippo Pathway: Upstream Signaling Antibody Sampler Kit
Brand: Cell Signaling Technology
Price: 514.00 USD
Availability: InStock

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Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 1 — 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).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 2 — 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 3 — Confocal immunofluorescent analysis of PC-3 cells using Merlin (D3S3W) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 4 — Western blot analysis of extracts from various cell lines using PTPN14 (D5T6Y) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 5 — CUT&RUN was performed with NCI-H2052 cells and YAP (D8H1X) XP^® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795. The figures show binding across TSPAN2, a known target gene of YAP (see additional figure containing CUT&RUN-qPCR data).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 6 — 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 using SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795. The figure shows binding across CTGF, a known target gene of YAP (see additional figure containing ChIP-qPCR data).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 7 — Western blot analysis of extracts from control HeLa cells (Lane 1) or YAP knockout HeLa cells (Lane 2) using YAP (D8H1X) XP^® Rabbit mAb (upper) or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the YAP knockout HeLa cells confirms the specificity of the antibody for YAP.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 8 — CUT&RUN was performed with NCI-H2052 cells and YAP (D8H1X) XP^® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795. The figures show binding across across chromosome 1 (upper), including TSPAN2 (lower), a known target gene of YAP (see additional figure containing CUT&RUN-qPCR data).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 9 — 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 using SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795. The figure shows binding across chromosome 6 (upper), including CTGF (lower), a known target gene of YAP (see additional figure containing ChIP-qPCR data).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 10 — 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 cells are negative for YAP protein expression.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 11 — CUT&RUN was performed with NCI-H2052 cells and either YAP (D8H1X) XP^® Rabbit mAb or Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human C1D downstream primers, human TSPAN2 upstream primers, and human ITM2A upstream primers. 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 13 — 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 14 — Immunohistochemical analysis of paraffin-embedded human non-Hodgkin’s lymphoma using YAP (D8H1X) XP^® Rabbit mAb performed on the Leica^® BOND™ Rx.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 15 — Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using YAP (D8H1X) XP^® Rabbit mAb performed on the Leica^® BOND™ Rx.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 16 — Immunohistochemical analysis of paraffin-embedded human breast adenocarcinoma using YAP (D8H1X) XP^® Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 17 — Immunohistochemical analysis of paraffin-embedded human breast adenocarcinoma using YAP (D8H1X) XP^® Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 18 — Immunohistochemical analysis of paraffin-embedded 293T cell pellet, wild-type (left, positive) or YAP/TAZ knockout (right, negative), using YAP (D8H1X) XP^® Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 19 — Immunohistochemical analysis of paraffin-embedded BEN cell pellet, untransfected (left, YAP/TAZ low), YAP-transfected (middle) or TAZ-transfected (right), using YAP (D8H1X) XP^® Rabbit mAb (top) or TAZ (E9J5A) XP^® Rabbit mAb #72804 (bottom).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 20 — Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma (left), colon carcinoma case 1 (middle) or colon carcinoma case 2 (right), using YAP (D8H1X) XP^® Rabbit mAb (top) or TAZ (E9J5A) XP^® Rabbit mAb #72804 (bottom).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 21 — Immunohistochemical analysis of paraffin-embedded mouse small intesine using YAP (D8H1X) XP^® Rabbit mAb (left) or YAP Antibody (right). These two antibodies detect unique, non-overlapping epitopes on YAP protein. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 22 — Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using YAP (D8H1X) XP^® Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 23 — Immunohistochemical analysis of paraffin-embedded PANC-1 (left) and RL (right) cell pellets using YAP (D8H1X) XP^® Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 24 — Immunohistochemical analysis of paraffin-embedded human benign prostatic hyperplasia using YAP (D8H1X) XP^® Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 25 — Confocal immunofluorescent analysis of low confluence MCF 10A cells (left), high confluence MCF 10A (center), and YAP negative RL 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 26 — Flow cytometric analysis of RL 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 27 — Western blot analysis of extracts from various cell lines using FRMD6 (D8X3R) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 28 — Western blot analysis of extracts from various cell lines using MST1 (D8B9Q) Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 29 — 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 30 — 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 31 — Western blot analysis of extracts from various cell lines using Ajuba (D4D8P) Rabbit mAb.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 32 — 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 33 — 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 using SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795. 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 34 — 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 35 — 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 using SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795. The figure shows binding across chromosome 1 (upper), including SMYD3 (lower), a known target gene of TAZ (see additional figure containing ChIP-qPCR data).

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 36 — Chromatin immunoprecipitations were performed with cross-linked chromatin from NCI-H2052 cells and either TAZ (D3I6D) Rabbit mAb or 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.

Hippo Pathway: Upstream Signaling Antibody Sampler Kit: Image 37 — 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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To Purchase # 56612T

Product #	Size	Price
56612T	1 Kit (7 x 20 microliters)	$ 514

Product Includes
Related Products

Product Includes	Quantity	Applications	Reactivity	MW(kDa)	Isotype
YAP (D8H1X) XP^® Rabbit mAb 14074	20 µl	WB IP IHC IF F ChIP C&R	H M R Hm Mk	65-78	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

Kit Usage Information

Protocols

7074: Western Blotting
12888: Western Blotting, Immunoprecipitation (Magnetic), Immunofluorescence*
13808: Western Blotting
14074: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Leica® Bond™), Immunohistochemistry (Paraffin), Immunofluorescence, Flow, ChIP Magnetic, Chromatin IP-seq, CUT&RUN Assay
14688: Western Blotting, Immunoprecipitation (Agarose)
14946: Western Blotting, Immunoprecipitation (Agarose)
34648: Western Blotting, Immunoprecipitation (Agarose)
70148: Western Blotting, Immunoprecipitation (Magnetic), ChIP Magnetic, Chromatin IP-seq

Product Description

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.

Specificity / Sensitivity

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

Source / Purification

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.

Background

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).

Pathways & Proteins

Explore pathways + proteins related to this product.

Database Links

UniProt ID: Q9GZV5 , P35240 , Q96NE9 , P46937 , Q15678 , Q96IF1 , Q13043

Entrez-Gene Id: 25937 , 4771 , 122786 , 10413 , 5784 , 84962 , 6789

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Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.

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Hippo Pathway: Upstream Signaling Antibody Sampler Kit #56612