To generate the construct V5-FZD5-CFP, V5-FZD5 was amplified by polymerase chain reaction (PCR) (forward primer: 5′-atctggtgggtcatcctgtc-3′; reverse primer: 5′-ctgatgtctagatacgtgcgacagggacacttg-3′) and subcloned into CFP-pcDNA3 between the Hind III and Xba I restriction sites. To create the FRET-based receptor sensors, the six amino acids of the FlAsH-binding sequence CCPGCC (5′-tgttgcccgggctgctgt-3′) were inserted within the third intracellular loop of FZD5 by standard overlapping PCR reactions. For the sensor V5-FZD5-FlAsH436-CFP, the motif was introduced between the amino acids Gly436 and Gly437 using the following forward primers (1 to 3) 5′-tgttgcccgggctgctgtggcactaagacggacaagcta-3′, 5′-agcgtcatcaagcagggttgttgcccgggctgctgtg-3′, and 5′-tcactcttccgcatccggagcgtcatcaagcagggttgt-3′ and the following reverse primer: 5′-ctgatgtctagatacgtgcgacagggacacttg-3′. For the sensor V5‑FZD5-FlAsH439-CFP, the FlAsH-binding motif was introduced between the amino acids Lys439 and Thr440 using the following forward primers (1 to 3): 5′-tgttgcccgggctgctgtacggacaagctagagaagctc-3′, 5′-aagcagggtggcactaagtgttgcccgggctgctgtacg-3′, and 5′-cgcatccggagcgtcatcaagcagggtggcactaagtgt-3′ and the following reverse primer: 5′-ctcactctagatacgtgcgacag-3′. To generate the constructs without CFP (V5-FZD5-FlAsH436 and V5-FZD5-FlAsH439), the region containing the FlAsH-binding motif was inserted into the V5-FZD5 plasmid between the Hind III and BstX I restriction sites. To create V5-FZD5-mCherry, a Bgl II site was cloned behind the Hind III site and an Age I site was placed in front of the Xba I restriction site in V5-FZD5-CFP. To add the Bgl II site, a forward primer (5′-cagtaagcttagatctaccatggtcccgtgcacgctg-3′) and a reverse primer (5′-gtgcgcaccttgttgtagag-3′) were used. To add the Age I site, the following forward and reverse primers were used: 5′-cagtgtcaagtccattacgg-3′ and 5′-agcagtaccggttgtacgtgcgacagggacacttg-3′, respectively. From this, V5-FZD5 was subcloned into pmCherry-N1 between the Bgl II and Age I restriction sites. To create SNAP-FZD5-Rluc8, SNAP-FZD5 was cloned into pRluc8-N1 using the following primers and inserted with Hind III and Age I restriction sites: forward, 5′-gacaagcttgccaccatggtcccgtgcacgctgctcctg-3′; reverse, 5′-cgtaccggtgctacgtgcgacagggacacttgcttgtggtatgc-3′.

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