Recombinant SHH was expressed in bacteria and purified in the lab as previously described (Bishop et al., 2009). Briefly, His-tagged SHH-N (C24II followed by human SHH amino acids 25-193) was expressed in Escherichia coli [BL21 strain; Rosetta2 (DE3)pLysS]. Cells were lysed in 10 mM phosphate buffer (pH 7.5), 500 mM NaCl, 1 mM 2-mercaptoethanol, 1 mM PMSF and 1× protease inhibitor cocktail, followed by centrifugation at 20,000 g for 30 min at 4°C. Clarified samples were incubated with Ni-NTA resin (Qiagen) for 1 h at 4°C. The resin was washed with 20 column volumes of wash buffer A (lysis buffer without protease inhibitors), followed by wash buffer B (wash buffer A+10 mM Imidazole), and bound proteins were eluted with elution buffer (wash buffer A+250 mM Imidazole). Peak fractions were pooled, concentrated using a 5 kDa cut-off VIVASPIN 15R (Life Technologies), and loaded onto a Superdex 75 gel filtration column (Amersham Biosciences) equilibrated with column buffer [10 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT]. The recombinant protein was >98% pure, as assessed from Coomassie staining, and stored at −80°C. The selection antibiotic puromycin was purchased from MilliporeSigma. The transfection reagent XtremeGENE 9 was purchased from Roche Molecular Systems and polybrene from MilliporeSigma. Bafilomycin A1 was purchased from Cayman Chemical. Vismodegib and Bortezomib were purchased from LC labs. The following primary antibodies were purchased from the following vendors: mouse anti-1D4 (The University of British Columbia, 56504; 1:5000); rat anti-E-cadherin (clone ECCD-2, Thermo Fisher Scientific, 13-1900; 1:1000); mouse anti-FLAG (clone M2, MilliporeSigma, F1804; 1:2000); goat anti-GFP (Rockland Immunochemicals, 600-101-215; 1:1000); rabbit anti-GFP (Novus Biologicals, NB600-308; 1:5000); mouse anti-GLI1 (clone L42B10, Cell Signaling, 2643; 1:1000); mouse anti-HA (clone 2-2.2.14, Thermo Fisher Scientific, 26183; 1:2000); rabbit anti-p38 (Abcam, ab7952; 1:2000); and rabbit anti-RNF156 (anti-MGRN1, Proteintech, 11285-1-AP; 1:500); mouse anti-ɑ-Tubulin (Clone DM1A, MilliporeSigma, T6199; 1:10,000); mouse anti-acetylated-Tubulin (MilliporeSigma, T6793; 1:10,000). The following primary antibodies were generated in the lab or received as a gift: guinea pig anti-ARL13B (1:1000) (Dorn et al., 2012); rabbit anti-SMO (designed against an intracellular epitope, 1:2000) (Rohatgi et al., 2007); and rabbit anti-MEGF8 (1:2000) (Kong et al., 2020). Hoechst 33342 and secondary antibodies conjugated to horseradish peroxidase (HRP) or Alexa Fluor dyes were obtained from Jackson ImmunoResearch Laboratories and Thermo Fisher Scientific as follows: Peroxidase AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 715-035-150, 1:10,000); Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-144, 1:10,000); Peroxidase AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, 705-035-003, 1:10,000); donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21206, 1:1000); donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed secondary antibody, Alexa Fluor 594 (Thermo Fisher Scientific, A-21207, 1:1000); donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202, 1:1000); donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 647 (Thermo Fisher Scientific, A-31571, 1:1000); Alexa Fluor 488 AffiniPure donkey anti-guinea Pig IgG (H+L) (Jackson ImmunoResearch Laboratories, 706-545-148, 1:1000); and Alexa Fluor 647 AffiniPure donkey anti-guinea pig IgG (H+L) (Jackson ImmunoResearch Laboratories, 706-605-148, 1:1000).