| Antibodies |
| rabbit monoclonal anti-SHH (clone C9C5) |
Cell Signaling Technology |
Cat# 2207S; RRID: AB_2188191 |
| rabbit monoclonal anti-WNT3A (clone C64F2) |
Cell Signaling Technology |
Cat# 2721; RRID: AB_2215411 |
| rabbit polyclonal anti-GFP |
Rockland |
Cat# 600-401-215; RRID: AB_828167 |
| mouse monoclonal anti-tubulin (clone DM1A) |
Sigma; Blose et al., 1984
|
Cat# T6199; RRID: AB_477583 |
| mouse monoclonal anti-acetylated tubulin (clone 6-11B-1) |
Sigma |
Cat# T7451; RRID: AB_609894 |
| goat polyclonal anti-mSMO |
Nedelcu et al., 2013 |
N/A |
| rabbit polyclonal anti-mCherry |
Nedelcu et al., 2013 |
N/A |
| chicken polyclonal anti-mARL13B |
This study |
N/A |
| rat anti-HA (clone 3F10)–HRP conjugate |
Roche |
Cat# 12013819001; RRID: AB_390917 |
| sheep anti-mouse IgG–HRP conjugate |
Jackson ImmunoResearch |
Cat# 515-005-003; RRID: AB_2340287 |
| donkey anti-rabbit IgG–HRP conjugate |
GE Healthcare |
Cat# NA934; RRID: AB_772206 |
| donkey anti-goat IgG–Alexa Fluor 488 conjugate |
Thermo |
Cat# A-11055; RRID: AB_2534102 |
| donkey anti-rabbit IgG–Alexa Fluor 555 conjugate |
Thermo |
Cat# A-31572; RRID: AB_162543 |
| donkey anti-chicken IgY–Alexa Fluor 647 conjugate |
Jackson ImmunoResearch |
Cat# 703-605-155; RRID: AB_2340379 |
| Bacterial and Virus Strains |
|
E. coli BL21 (DE3) pLysS |
Sigma |
Cat# 69451 |
| Chemicals, Peptides, and Recombinant Proteins |
| polyethylenimine (PEI), linear, MW 25000 |
Polysciences |
Cat# 23966 |
|
TransIT®-293 Transfection Reagent |
Mirus |
Cat# MIR2700 |
| cycloheximide (≥94%) |
Sigma |
Cat# C7698 |
| methyl-β-cyclodextrin (MβCD) |
Sigma |
Cat# 332615 |
| IWP-2 (PORCN inhibitor) (≥98%) |
Sigma; Wang et al., 2013
|
Cat# I0536 |
| SAG (SMO agonist) (≥98%) |
Axxora; Chen et al., 2002
|
Cat# BV-1939 |
| SANT-1 (SMO antagonist) (≥95%) |
Calbiochem; Chen et al., 2002
|
Cat# 559303 |
| gramicidin A |
Sigma |
Cat# 50845 |
| veratridine (≥98%) |
Tocris Bioscience |
Cat# 2918 |
| bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) (≥95%) |
Sigma |
Cat# D8189 |
| carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (≥97%) |
Sigma |
Cat# C2759 |
| staurosporine (>99%) |
LC Laboratories |
Cat# S-9300 |
| oligomycin A (≥99%) |
Sigma |
Cat# 75351 |
| See Table S1 for composition of modified Tyrode’s media utilized in this study. |
This study |
N/A |
| FLAG peptide: NH2-DYKDDDDK-OH |
Genscript |
N/A |
| 6xHis-mARL13B (antigen for immunization) |
This study |
N/A |
| MBP-mARL13B (for antibody affinity purification) |
This study |
N/A |
| FLAG-HT7-mSCUBE2-ΔFurin |
This study |
N/A |
| Critical Commercial Assays |
| MiSeq Reagent Kit v2 |
Illumina |
Cat# MS-102-2001 |
| Affi-Gel 10 Gel |
Bio-Rad |
Cat# 1536099 |
| anti-FLAG M1 resin |
Millipore |
Cat# A4596 |
| Amicon Ultra-4 Centrifugal Filter Unit – 10kDa cutoff |
Millipore |
Cat# UFC8010 |
| Superdex 200, 10/300 GL |
GE Healthcare |
Cat# 17517501 |
| Nano-Glo® Luciferase Assay |
Promega |
Cat# N1120 |
| BCA Assay |
G Biosciences |
Cat# 786-570 |
| TRIzol Reagent |
Thermo |
Cat# 15596018 |
| RQ1 RNase-Free DNase |
Promega |
Cat# M6101 |
| LunaScript RT SuperMix Kit |
NEB |
Cat# E3010L |
| PowerUp SYBR Green Master Mix |
Thermo |
Cat# A25742 |
| CellTiter-Glo® Luminescent Cell Viability Assay |
Promega |
Cat# G7572 |
| MTT Cell Proliferation Assay Kit |
Cayman Chemical |
Cat# 10009365 |
| Experimental Models: Cell Lines |
| Human: HEK293T |
ATCC |
Cat# CRL-3216; RRID: CVCL_0063 |
| Human: HEK293S GnTI−
|
A.C. Kruse; ATCC; Reeves et al., 2002
|
Cat# CRL-3022; RRID: CVCL_A785 |
| Mouse: L cell |
ATCC |
Cat# CRL-2648; RRID: CVCL_4536 |
| Mouse: L Wnt-3A |
ATCC |
Cat# CRL-2647; RRID: CVCL_0635 |
| Mouse: NIH-3T3 |
ATCC |
Cat# CRL-1658; RRID: CVCL_0594 |
| Mouse: Ptch1
−/− MEF |
Goodrich et al., 1997 |
N/A |
| Human: DISP1KO HEK293T |
This study |
N/A |
| Oligonucleotides |
| mRpl27 qRT-PCR forward primer: 5’-GTCGAGATGGGCAAGTTCAT-3’ |
Nedelcu et al., 2013 |
N/A |
| mRpl27 qRT-PCR reverse primer: 5’-GCTTGGCGATCTTCTTCTTG-3’ |
Nedelcu et al., 2013 |
N/A |
| mGli1 qRT-PCR forward primer: 5’-GGCCAATCACAAGTCAAGGT-3’ |
Nedelcu et al., 2013 |
N/A |
| mGli1 qRT-PCR reverse primer: 5’-TTCAGGAGGAGGGTACAACG-3’ |
Nedelcu et al., 2013 |
N/A |
| See Table S2 for a list of CRISPR gRNA oligonucleotides and sequencing primers utilized in this study. |
This study |
N/A |
| Recombinant DNA |
| See Table S3 for a list of all plasmids utilized in this study. |
This study |
N/A |
| Software and Algorithms |
| MetaMorph Microscopy Automation and Image Analysis Software |
Molecular Devices |
http://www.moleculardevices.com/Products/Software/Meta-Imaging-Series/MetaMorph.html; RRID: SCR_002368 |
| MATLAB |
MathWorks |
http://www.mathworks.com/products/matlab/; RRID: SCR_001622 |
| FIJI |
National Institutes of Health |
http://fiji.sc; RRID: SCR_002285 |
| JalView |
Waterhouse et al., 2009 |
http://www.jalview.org/; RRID: SCR_006459 |
| PyMOL |
Schrodinger |
http://www.pymol.org/; RRID:SCR_000305 |
| Prism 8 |
GraphPad |
http://www.graphpad.com/; RRID: SCR_002798 |
| Photoshop CS5 |
Adobe |
https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 |
| Inkscape |
Inkscape |
https://inkscape.org/en/; RRID: SCR_014479 |
