Generation of Bioluminescent Fusion Proteins

To generate HiBiT-SMO and ΔCRD HiBiT-SMO, Nluc sequence in Nluc-SMO or ΔCRD Nluc-SMO (coding mouse SMO, ref. ^{26 (link)}) were replaced with HiBiT sequence (nucleotides sequence: 5′-GTG AGC GGC TGG CGG CTG TTC AAG AAG ATT AGC-3′; amino acids sequence: VSGWRLFKKIS). To generate FLAG-SNAP-SMO, mouse SMO sequence from SMO-Rluc8 was inserted into an empty FLAG-SNAP-tagged pcDNA3.1 vector between BamHI and HindIII sites. SMO-Rluc8, HiBiT-FZD₆, SNAP-FZD₄, SNAP-FZD₅, SNAP-FZD₆, SNAP-FZD₇, FZD₄-Nluc, FZD₆-Nluc, β₂AR, Venus-KRas, and Nluc-DVL2 were generated and validated in our previous studies^{16 (link),29 (link)}. SMO-Nluc and the G_s BRET sensor were generated using prolonged overlap extension PCR techniques. The plasmid encoding CD86-Nluc was provided by Dr. Ulrike Zabel (University of Wuerzburg, Wuerzburg, Germany). Plasmids encoding cPKA-YFP and NbSmo2-YFP were a kind gift from Benjamin Myers (University of Utah, Salt Lake City, USA)⁴⁴ . Plasmid encoding Venus-mGsi was from Nevin Lambert (Augusta University, Georgia, USA^{75 (link)}). Wild-type human H₃R DNA vector was purchased from cDNA.org. The desired mutations were generated using GeneArt site-directed mutagenesis kit (Thermo Fisher Scientific). All constructs were validated by sequencing (Eurofins GATC, Konstanz, Germany). Utilized primers are listed in Supplementary Table 1.