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26118
Hedgehog Signaling Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Hedgehog Signaling Antibody Sampler Kit #26118

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Hedgehog Signaling Antibody Sampler Kit: Image 1
Immunoprecipitation of GLI2 protein from SNU-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is GLI2 (E5V8N) Rabbit mAb. Western blot analysis was performed using GLI2 (E7R1N) Rabbit mAb #66670. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Hedgehog Signaling Antibody Sampler Kit: Image 2
Western blot analysis of extracts from various cell lines using GLI2 (E5V8N) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). As expected, BEN cells are negative for GLI2 expression.
Hedgehog Signaling Antibody Sampler Kit: Image 3
Western blot analysis of extracts from 293T cells, mock transfected (lane 1) or transiently transfected with plasmid encoding Myc/DDK-tagged GLI2 protein (lane 2), using GLI2 (E5V8N) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Hedgehog Signaling Antibody Sampler Kit: Image 4
Western blot analysis of extracts from HT29 and GH3 cells, and GH3 cell conditioned medium (CM), using Shh (C9C5) Rabbit mAb.
Hedgehog Signaling Antibody Sampler Kit: Image 5
Western blot analysis of total cell lysates from COS cells, untransfected or transiently transfected with a human PTCH1 expression construct, using PTCH1 (C53A3) Rabbit mAb.
Hedgehog Signaling Antibody Sampler Kit: Image 6
Western blot analysis of extracts from COS cells, untransfected or transiently transfected with a construct expressing human PTCH2, using PTCH2 (G1191) Antibody.
Hedgehog Signaling Antibody Sampler Kit: Image 7
Western blot analysis of total cell lysates from various cell types using SUFU (C54G2) Rabbit mAb.
Hedgehog Signaling Antibody Sampler Kit: Image 8
Western blot analysis of extracts from RMS-13 and TOV-112D cells using GLI1 (C68H3) Rabbit mAb.
Hedgehog Signaling Antibody Sampler Kit: Image 9
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.
Hedgehog Signaling Antibody Sampler Kit: Image 10
Western blot analysis of extracts from various cell lines using GLI3 (E6E2K) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Low expression of GLI3 protein in NCI-H146 and Hep G2 cells is consistent with predicted expression patterns, confirming specificity of the antibody for GLI3.
Hedgehog Signaling Antibody Sampler Kit: Image 11
Immunoprecipitation of Smoothened protein from extracts of 293T cells transiently transfected with plasmid encoding Myc/DDK-tagged Smoothened protein. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Smoothened (E6Z5T) Rabbit mAb. Western blot analysis was performed using Smoothened (E6Z5T) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Hedgehog Signaling Antibody Sampler Kit: Image 12
Western blot analysis of extracts from 293T cells, mock transfected (lane 1) or transiently transfected with plasmid encoding Myc/DDK-tagged Smoothened protein (lane 2), using Smoothened (E6Z5T) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). *The antibody detects 25 kDa and 38 kDa proteins of unknown identity in some cell extracts.
Hedgehog Signaling Antibody Sampler Kit: Image 13
Immunoprecipitation of endogenous PTCH1 from 293 cell lysates using PTCH1 (C53A3) Rabbit mAb. Western blot detection was performed using the same antibody.
Hedgehog Signaling Antibody Sampler Kit: Image 14
Immunoprecipitation of GLI3 protein from RD cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is GLI3 (E6E2K) Rabbit mAb. Western blot analysis was performed using GLI3 (E6E2K) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
To Purchase # 26118
Cat. # Size Qty. Price
26118T
1 Kit  (8 x 20 microliters)
$ 592

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Shh (C9C5) Rabbit mAb 2207 20 µl
  • WB
H R 19, (45 precursor) Rabbit IgG
Smoothened (E6Z5T) Rabbit mAb 92981 20 µl
  • WB
  • IP
H 85 Rabbit IgG
PTCH1 (C53A3) Rabbit mAb 2468 20 µl
  • WB
  • IP
H 180-210 Rabbit IgG
PTCH2 (G1191) Antibody 2470 20 µl
  • WB
  • IP
H 130 Rabbit 
SUFU (C54G2) Rabbit mAb 2520 20 µl
  • WB
H M R Mk 54 Rabbit IgG
GLI1 (C68H3) Rabbit mAb 3538 20 µl
  • WB
  • IP
H 160 Rabbit IgG
GLI2 (E5V8N) Rabbit mAb 18773 20 µl
  • WB
  • IP
H M Mk 220 Rabbit IgG
GLI3 (E6E2K) Rabbit mAb 71107 20 µl
  • WB
  • IP
H 200 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Hedgehog Signaling Antibody Sampler Kit provides an economical means of evaluating key members of the Hedgehog signaling pathway. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Shh (C9C5) Rabbit mAb detects endogenous levels of total Shh protein. This antibody does not cross-react with transfected IHH or DHH. PTCH1 (C53A3) Rabbit mAb detects transfected levels of PTCH1. This antibody can also detect endogenous levels of PTCH1 through immunoprecipitation followed by western blot analysis. PTCH2 (G1191) Antibody detects transfected levels of PTCH2 protein. It does not recognize transfected levels of human PTCH1 protein. Smoothened (E6Z5T) Rabbit mAb detects transfected levels of total smoothened (SMO) protein. SUFU (C54G2) Rabbit mAb detects endogenous levels of total SUFU protein. GLI1 (C68H3) Rabbit mAb detects endogenous levels of total GLI1 protein. GLI2 (E5V8N) Rabbit mAb detects endogenous levels of total GLI2 protein. GLI3 (E6E2K) Rabbit mAb detects endogenous levels of total GLI3 protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly1191 of human PTCH2. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Glu53 of human Shh, Pro1307 of human PTCH1, Leu458 of human SUFU, Gly420 of human GLI1, and Pro1188 of human GLI2 protein, or recombinant proteins corresponding to human smoothened (SMO) protein, and the carboxy terminus of human GLI3.

Background

The Hedgehog (Hh) signaling pathway plays critical roles in the regulation of cell fate, tissue patterning, and growth during embryonic development. It is downregulated during postnatal development, but can be reactivated to promote tissue repair and regeneration. Aberrant Hh signaling activity during prenatal development is associated with numerous birth defects (e.g., holoprosencephaly), while uncontrolled Hh pathway activation postnatally is linked to the development of several cancer types (1,2). There are three canonical Hh ligands: Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH), all of which have distinct as well as overlapping roles and expression patterns (3-5). Patched1 and 2 (PTCH1 and PTCH2) are partially redundant 12-pass transmembrane proteins that function as receptors for Hh ligands (6-8). Smoothened (SMO) is a 7-pass transmembrane G protein-coupled receptor (GPCR) that functions as the key transducer of Hh signaling. In the absence of Hh ligands (off-state), PTCH proteins are localized to cilia, and function to suppress SMO activity (1,2). Suppressor of Fused (SUFU) simultaneously contributes to suppression of the pathway by sequestering the glioma-associated oncogene (GLI) family of transcription factors (9,10). Binding of Hh ligands to PTCH receptors results in derepression of SMO, in part by promoting its translocation to cilia; this leads to downregulation of SUFU, resulting in the stabilization and nuclear translocation of GLI transcription factors that regulate the transcription of genes involved in cell proliferation, migration, and survival (1).
  1. Lee, R.T. et al. (2016) Development 143, 367-72.
  2. McMahon, A.P. et al. (2003) Curr Top Dev Biol 53, 1-114.
  3. Zhang, X.M. et al. (2001) Cell 106, 781-92.
  4. Adolphe, C. et al. (2004) Development 131, 5009-19.
  5. Pathi, S. et al. (2001) Mech Dev 106, 107-17.
  6. Stone, D.M. et al. (1996) Nature 384, 129-34.
  7. Motoyama, J. et al. (1998) Nat Genet 18, 104-6.
  8. Smyth, I. et al. (1999) Hum Mol Genet 8, 291-7.
  9. Barnfield, P.C. et al. (2005) Differentiation 73, 397-405.
  10. Méthot, N. and Basler, K. (2000) Development 127, 4001-10.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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