Heterozygous mutations alter Wnt pathway activities in HEK293T cells. (A,C,E) HEK293T cells were cotransfected with Wnt1 expression plasmid (amounts indicated), wild-type (WT), or mutant (MT), or equal amounts of WT and MT plasmids, along with the reporter plasmids SuperTOPflash and pRL-TK. Forty-eight hours after transfection, luciferase activity was measured from 3 independent experiments. All WT, MT, or WT/MT plasmids were introduced at 0.1 ng, 0.1 ng, 50 ng, and 5 ng for BCL9, DKK2, CSNK1E, and FZD5, respectively. LRP6 plasmid (10 ng) was also included in the FZD5 mutation characterization. MT1: Y46*; MT2: V290I. Downstream Wnt pathway targets were also assessed for mutated DKK2 by gene expression (see supplemental Figure 7 and supplemental Methods). (B) HEK293T cells were cotransfected with 20 or 80 ng of WT or MT RYK along with reporter plasmids. At 24 hours after the transfection, recombinant Wnt3a was added (25 ng/mL final concentration) and incubated for 24 hours before luciferase activity was measured. Detection of phosphorylation of downstream target DVL2 was assessed by western blot analysis (see supplemental Figure 7 and supplemental Methods). (D) HEK293T cells were cotransfected with either WT or MT WNT1 along with the reporter plasmids. Luciferase activity was measured from three independent experiments 48 hours after transfection. For more details on the conditions of Wnt activation, please see supplemental Methods.