Fzd1/2/4/5/7/8-knockout HEK293T cells (69 (link)) were maintained in DMEM supplemented with 10% v/v fetal bovine serum. Cells were seeded into white 96-well plates (PerkinElmer) and transfected with 80 ng SuperTopFlash plasmid (laboratory of Dr. Randall Moon; Addgene plasmid #12456), 20 ng CMV-driven LacZ plasmid, and 1 ng FLAG-tagged Fzd4 or FZD5 plasmid per well using Lipofectamine 2000 according to the manufacturer’s instructions (ThermoFisher). Purified xWnt8 or Norrin (with MBP tag removed as described above for BLI) were added 24 h post-transfection and cells were incubated for 16–18 h with ligand. Cells were lysed and luciferase and β-galactosidase signals were quantified on a BioTek Synergy 2 plate reader using the Dual-Light Reporter System (ThermoFisher) according to the manufacturer’s instructions.