Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: SHH , RnD_Systems_Own , 1314-SH-025 , Ligand.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Sonic Hedgehog repression underlies gigaxonin mutation–induced motor deficits in giant axonal neuropathy

doi: 10.1172/JCI129788

Figure Lengend Snippet: (A) NIH-3T3 cells were transfected with human Cherry-Gig plasmid ± 3 μg/mL Shh for 48 hours. Degradation of endogenous Ptch protein occurs only in the presence of both ectopic gigaxonin and Shh. (B) Repression of the endogenous gigaxonin using siRNA causes an increase of the endogenous Ptch levels in Shh Light-2 cells, which is potentiated by Shh induction. (C and D) Interaction between gigaxonin and Ptch, as revealed by reverse immunoprecipitation on both endogenous (C) and ectopic (D) Ptch protein. Shh Light-2 cells transfected with human Cherry-Gig (C); COS-7 cells transfected with zebrafish 3Flag-Gig and zebrafish Cherry-Ptch (D). IgG serves as an internal negative control. (D) Cherry immunoprecipitation identifies Ptch proteins, which are mainly not modified, while the Ptch pool enriched in gigaxonin immunocomplex presents a laddering overlapping with K48-specific ubiquitin chain. (E) Model of action of the gigaxonin–E3 ligase in the initiation of Shh signaling. In an off state (left), prior to Shh activation, receiving cells silence the cascade through the inhibitory effect of the Ptch receptor on the effector Smo. Upon Shh production, the active Shh form is released and received by progenitor cells. Gigaxonin acts as an initiator of signal transduction by degrading Shh-bound Ptch receptor (middle), hence allowing the derepression of the signal transducer Smo, which translocates into the cilium to activate the pathway. In the absence of gigaxonin (right), receiving tissues are unable to interpret Shh signaling, due to the constitutive repression of Smo induced by the accumulation of Shh-bound Ptch receptor.

Article Snippet: At 24 hours after transfection, NIH-3T3 cells were treated with 3 μg/mL ShhN (1314-SH, R&D Systems) ( 39 ) and Light-2 cells with Shh-conditioned medium (Shh-CM) obtained from ShhN-expressing HEK293 cells for 48 hours (provided by P. Beachy, GRCF Biorepository & Cell Center, Johns Hopkins University, Baltimore, Maryland, USA) ( 64 ).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control, Modification, Activation Assay, Transduction

Journal: Nature chemical biology

Article Title: The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism

doi: 10.1038/s41589-019-0370-y

Figure Lengend Snippet: a, Schematic of the various SHH proteins used in signalling assays. b, Two different amounts of each SHH variant from (h) were analyzed by immunoblotting. Full gels can be found on Supplementary Figure 7. c and d, Fold-increase in Gli1 mRNA (relative to the no SHH added condition) was used as a metric for signalling strength after treatment of NIH/3T3 cells with various concentrations of the indicated ligands. e, HH signalling strength at increasing concentrations of the N-terminal palmitoylated peptide (Palm-ShhN15) in the absence or presence (100 μM) of a cholesteroylated C-terminal peptide (ShhN7-chol). In c-e, the mean is depicted and error bars reflect standard deviation (n=4). Each experiment in c-e was repeated at least 3 times.

Article Snippet: A rabbit monoclonal antibody (clone C9C5) against the N-terminal signaling domain of human SHH was obtained from Cell Signaling Technologies (cat#2207).

Techniques: Variant Assay, Western Blot, Standard Deviation