Figure 3. Antagonist effects of Lgr4-ED.

(A) For the R-spondin assays, HEK293T cells were transfected with plasmids encoding TOPFLASH, CMV-β-Gal and human LGR4. At 24 h after transfection, cells were further treated with WNT3A, RSPO2 and graded amounts of purified Lgr4-ED as indicated. (B) For the norrin assays, HEK293T cells were co-transfected with TOPFLASH, CMV-β-Gal, LGR4, norrin and increasing amounts of rat Lgr4-ED plasmids as indicated. After recovery in serum-containing medium for 24 h, the cells were incubated in the serum-free medium for 16 h before performing luciferase assays. Luciferase data were normalized against the β-galactosidase levels and are shown as the mean ± SD of triplicates (*, P<0.05). Three additional experiments were performed and all three produced similar results.