Cell Signaling Technology

Product Pathways - Development

Hedgehog Signaling Antibody Sampler Kit #8358

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Shh (C9C5) Rabbit mAb #2207 40 µl W H R Z (M) 19, (45 kDa precursor) Rabbit IgG
PTCH1 (C53A3) Rabbit mAb #2468 40 µl W IP H 180-210 Rabbit IgG
PTCH2 (G1191) Antibody #2470 40 µl W IP H 130 Rabbit
SUFU (C54G2) Rabbit mAb #2520 40 µl W IP H M R Mk 54 Rabbit IgG
GLI1 (V812) Antibody #2534 40 µl W IP H (M) (R) 160 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Z=Zebrafish
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Shh (C9C5) Rabbit mAb detects endogenous levels of total Shh protein. This antibody does not cross-react with transfected IHH and 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. SUFU (C54G2) Rabbit mAb detects endogenous levels of total SUFU protein. GLI1 (V812) Antibody detects endogenous levels of total GLI1 protein.

Western blot analysis of extracts from HT29 and GH3 cells, and GH3 cell conditioned medium (CM), using Shh (C9C5) Rabbit mAb #2207.

Western blot analysis of total cell lysates from COS cells, untransfected or transiently transfected with a human PTCH1 construct, using PTCH1 (C53A3) Rabbit mAb #2468.

Western blot analysis of extracts from COS cells, untransfected or transiently transfected with a construct expressing human PTCH2, using PTCH2 (G1191) Antibody #2470.


Western blot analysis of total cell lysates from various cell types using SUFU (C54G2) Rabbit mAb #2520.

Western blot analysis of extracts from RMS-13 and TOV-112D cells using GLI1 (V812) Antibody #2534.

Description

This sampler kit provides an economical means of evaluating key members of the Hedgehog signaling pathway. The kit contains enough primary and secondary antibody to perform four western miniblot experiments.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region (predicted to be intracellular) surrounding Gly1191 of human PTCH2 or to residues surrounding Val812 of human GLI1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu53 of human Shh, Pro1307 of human PTCH1, or Leu458 of human SUFU.

Background

The evolutionarily conserved Hedgehog (Hh) signaling pathway plays critical roles in the regulation of patterning, growth, and cell migration during embryonic development and adult tissue homeostasis. Aberrant Hh signaling activity can be associated with numerous birth defects and uncontrolled Hh pathway activation is linked to the development of several types of cancers (1-2). The three identified vertebrate Hh genes are Sonic (Shh), Indian (Ihh), and Desert (Dhh), all of which have distinct as well as overlapping roles (3-5). Patched1 and 2 (PTCH1 and PTCH2) are twelve-pass transmembrane proteins that function as the Hh receptors (6-9). The general organization of the Hh pathway consists of a series of repressive interactions. In the absence of Hh proteins (off-state), PTCH suppresses the otherwise constitutively active signaling receptor Smoothened (Smo) (1,2). In the off-state, SUFU (Suppressor of Fused), originally identified in Drosophila as a suppressor of the Fused (Fu) kinase (10), suppresses Hh signaling by regulating the localization of the transcription factors Gli and Ci (11,12). In Drosophila, SUFU may also positively regulate Hh signaling depending on SUFU protein levels and Hh signal intensity (13).

  1. Ingham, P.W. and McMahon, A.P. (2001) Genes Dev 15, 3059-87.
  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. Chen, Y. and Struhl, G. (1996) Cell 87, 553-63.
  8. Motoyama, J. et al. (1998) Nat Genet 18, 104-6.
  9. Smyth, I. et al. (1999) Hum Mol Genet 8, 291-7.
  10. Pham, A. et al. (1995) Genetics 140, 587-98.
  11. Barnfield, P.C. et al. (2005) Differentiation 73, 397-405.
  12. Méthot, N. and Basler, K. (2000) Development 127, 4001-10.
  13. Dussillol-Godar, F. et al. (2006) Dev Biol 291, 53-66.

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