TOPflash Wnt reporter assays

L cells stably transfected with TOPflash dual reporter plasmid system (James Chen, Stanford University) were used in TOPflash dual luciferase assays (Promega Dual Luciferase kit) with Wnt3A conditioned medium from a stably transfected Wnt3A-expressing cell line (Roel Nusse, Stanford University) or recombinant Wnt3A (R&D). Recombinant murine Rspo1-4 (R&D) were used at 5 pM concentration each in these assays. Recombinant LGR5, Rnf43 and Znrf3 ECD proteins were expressed and purified as above and their purity and protein concentrations were determined by Coomassie-stained SDS-PAGE and Bradford assays. Assays were visualized with a Tecan M1000 luminometer. Recombinant scFv-DKK1c was expressed and purified per Janda et al.²⁵