Figure 7.

A well-conserved triad of cysteines in the linker domain is crucial for receptor function. A, schematic of the Cys-to-Ala mutations in FL FZD₆ (top) and ΔCRD-FZD₆ (bottom). Cys-161, Cys-181, and Cys-185 were individually mutated in both receptor constructs. B, confocal images of surface expression of FZD₆ Cys-to-Ala constructs, which were transiently transfected into HEK293T cells and detected with indirect immunofluorescence using a fluorophore-conjugated secondary antibody against the anti-FLAG antibody on nonpermeabilized cells (FLAG surface). FL FZD₆ was used as a positive control. C, surface expression was quantified using a cell ELISA based on detection of anti-FLAG under nonpermeabilized conditions. The scatter dot plot represents mean with S.D. of three independent experiments performed in triplicates. ***, p < 0.001. D, cell lysates were analyzed for the electrophoretic mobility shift of DVL2 by immunoblotting using anti-DVL2, anti-FZD₆, and anti-α-tubulin (loading control) antibodies. E, photomicrographs of the DVL2 recruitment assay performed on HEK293T cells transfected with Cys-to-Ala mutants and DVL2-HA and detected with anti-FLAG (red, FZD₆) and anti-HA (green, DVL2) antibody. Scale bars = 10 μm.