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.
Western blot analysis of extracts from HT29 and GH3 cells, and GH3 cell conditioned medium (CM), using Shh (C9C5) Rabbit mAb.
Immunoprecipitation of endogenous PTCH1 from 293 cell lysates using PTCH1 (C53A3) Rabbit mAb. Western blot detection was performed using the same antibody.
Western blot analysis of extracts from COS cells, untransfected or transiently transfected with a construct expressing human PTCH2, using PTCH2 (G1191) Antibody.
Western blot analysis of total cell lysates from various cell types using SUFU (C54G2) Rabbit mAb.
Western blot analysis of extracts from RMS-13 and TOV-112D cells using GLI1 (C68H3) Rabbit mAb.
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.
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.
|Shh (C9C5) Rabbit mAb 2207||20 µl||
||H R||19, (45 precursor)||Rabbit IgG|
|PTCH1 (C53A3) Rabbit mAb 2468||20 µl||
|PTCH2 (G1191) Antibody 2470||20 µl||
|SUFU (C54G2) Rabbit mAb 2520||20 µl||
||H M R Mk||54||Rabbit IgG|
|GLI1 (C68H3) Rabbit mAb 3538||20 µl||
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
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 two western miniblot experiments.
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 (C68H3) Rabbit mAb detects endogenous levels of total GLI1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region (predicted to be intracellular) surrounding Gly1191 of human PTCH2. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu53 of human Shh, Pro1307 of human PTCH1, Leu458 of human SUFU, or Gly420 of human GLI1.
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).
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