Figure 6. Analysis of mutations in FZD4 on its biological activity in the Norrin/β-catenin signaling pathway. A: SuperTopFlash (STF) cells/well were cotransfected with 700 ng DNA (200 ng of Norrin, 200 ng of LRP5, 100 ng of pSV-β-galactosidase control vector, and 200 ng of FZD4 [wild-type or mutation]) and 1.1 μl Lipofectamine 2000 transfection reagent. Forty-eight hours after transfection, the cells were harvested and washed twice with PBS. Luciferase activities were measured with a dual-luciferase assay kit. Reporter activity was normalized to the coexpressed β-galactosidase activity in each well. The positive control (LRP+/FZD4+/Norrin+) was normalized to 1. Each test was performed in triplicate. The reporter assay was repeated three times, and a representative result was obtained. Statistical analyses were performed with a two-tailed unpaired Student t test. B: Human embryonic kidney (HEK) 293 cells were transfected with 600 ng FLAG-FZD4 WT or mutant plasmids, respectively. Forty-eight hours post-transfection, cell lysates were immunoblotted (IB) with FLAG or actin. P, positive control. C: Protein quantification: the gray value of FLAG-FZD4/actin and normalized wild-type to 1 (1#:c.1188_1192del/p.F396fs; 2#: c.1220delC/p.A407Vfs; 3#: c.905G>A/p.C302Y; 4#: c.1325T>A/p.V442E).