Endogenous RYK and MIB1 are required for Wnt/CTNNB1 signaling. (A and C) Graphs showing results from a Wnt/CTNNB1-activated luciferase-based reporter (BAR). Data are mean + SD (error bars). (B and D–F) Quantitative RT-PCR data showing a requirement for RYK and MIB1 in the expression of Wnt target genes in multiple cell lines. Data are mean + SD (error bars). (A) HEK293T cells were transfected with siRNAs for 2 d and then treated overnight with control or Wnt-3A–conditioned media. Results are normalized to one for unstimulated cells. Multiple nonoverlapping siRNAs targeting either RYK or MIB1 significantly reduce Wnt-3A–mediated reporter activation. Data are representative of n > 5 experiments. (B) Increases in AXIN2 mRNA levels resulting from treatment with Wnt-3A–conditioned media are reduced in the context of MIB1 or RYK siRNA transfection in HEK293T cells. (C) Graph showing that MIB1 and RYK are required for Wnt-3A–mediated activation of BAR in U2OS cells treated as in A. n = 5. (D, E, and F) Graphs showing the results of quantitative RTPCR for AXIN2 (D and F) and NKD1 (E), demonstrating a requirement for MIB1 and RYK for the activation of endogenous Wnt-target genes by Wnt-3a in U2OS and HeLa cells. n = 3.