Figure 3.

F328S Frizzled-4 exhibits reduced Norrin-stimulation and Dvl2 recruitment. (A) Cell surface receptor expression detected by IFA in HEK293 cells contransfected with the amount in ng of V5mFz4 variant shown in parentheses and 1 ng of hLRP5 per well. Following fixation the nonpermeabilized cells were incubated with V5 primary antibody followed by an incubation with a compatible alexaflour594 secondary antibody and subsequent fluorescence quantification on a multimode plate reader. The dotted line represents the background fluorescence level of the wells transfected with hLRP alone. (B) V5mFz4 WT and F328S were overexpressed in HEK293 cells and their ability to induce Lef/Tcf-dependent luciferase enzyme induction following the addition of recombinant Norrin was performed as described in the Materials and Methods with an exception being that the wells for the “5X” hLRP5 condition were transfected with 5 ng of hLRP5 instead of 1 ng. (C) Confocal images of permeabilized HeLa cells cotransfected with GFP-tagged Dvl2 and V5mFz4WT or F328S using a V5 primary antibody.