Molecular Components of the Hedgehog Signaling Pathway

NIH 3T3 and 293T cells were obtained from ATCC, Smo−/− fibroblasts have been described previously^{33 (link)} and were originally obtained from Drs. James Chen and Philip Beachy, SAG was from Enzo Life Sciences, Cyclopamine from Toronto Research Chemicals, SANT-1 from EMD Millipore, 20(S)-OHC from Steraloids. The synthesis of 20(R)-OHC^{7 (link)} and 20(S)-amine coupled beads for SMO binding assays has been described in detail previously^{10 (link)}. Rabbit polyclonal antibodies against SMO, PTCH1 and SUFU have been described previously^{34 (link),35 (link)}, and the mouse monoclonal antibody against GLI1 was obtained from Cell Signaling Technologies (Cat#L42B10). SHH-containing conditioned media was made from 293T cells transfected with the N-terminal signalling domain of SHH and used at saturating concentrations (dilution of 1:4)^{2 (link)}.

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Byrne E.F., Sircar R., Miller P.S., Hedger G., Luchetti G., Nachtergaele S., Tully M.D., Mydock-McGrane L., Covey D.F., Rambo R.P., Sansom M.S., Newstead S., Rohatgi R, & Siebold C. (2016). Structural basis for Smoothened regulation by its extracellular domains. Nature, 535(7613), 517-522.

Publication 2016

Cyclopamine SANT-1 20(S)-OHC

293t cells Amine Antibodies Assays Conditioned media Cyclopamine Dilution Fibroblasts Monoclonal antibody Mouse Nih 3t3 Ptch1 Rabbit Synthesis

Corresponding Organization :

Other organizations : Wellcome Centre for Human Genetics, University of Oxford, Stanford University, Diamond Light Source, Washington University in St. Louis

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Variable analysis

independent variables

Smo knockout (Smo-/- fibroblasts)
Small molecule treatments (SAG, Cyclopamine, SANT-1, 20(S)-OHC, 20(R)-OHC)

dependent variables

Hedgehog signaling response (not explicitly mentioned)

control variables

NIH 3T3 cells
293T cells
SHH-containing conditioned media

controls

Positive control: SHH-containing conditioned media
Negative control: Not explicitly mentioned

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