MIB1 requires endogenous RYK to activate Wnt/CTNNB1 signaling. (A) Graph demonstrating that MIB1 cDNA overexpression but not catalytically impaired MIB1-C997S can activate BAR in the presence or absence of Wnt-3A–conditioned media. Cells were transfected 6–8 h before treatment with conditioned media overnight. Data are mean + SD. n = 3. (B) Data generated from quantitative RT-PCR or HEK293T cells overexpressing MIB1 showing activation of the Wnt target gene, AXIN2. Data are mean + SD (error bars). *, P < 0.05 using a Student’s t test. n = 3. (C) Western blot of HEK293T cells overexpressing MIB1 showing that MIB1 can activate LRP6 phosphorylation and stimulate CTNNB1 stabilization. In rows labeled conA, cell lysates were stripped of membrane-associated proteins by overnight incubation with conA Sepharose resin. Blot represents n = 3. (D) Data showing the enhancement of Wnt-3A–induced BAR activity by the overexpression of MIB1 but not by mutants of MIB1 that lack the ability to bind RYK or lack enzymatic activity. The dotted line indicates the level of reporter activation by Wnt-3A–conditioned media alone. Data are mean + SD (error bars). n = 3. (E) Data demonstrating that MIB1-mediated activation of BAR is reversed by multiple independent siRNAs targeting either CTNNB1 or RYK. The dotted line indicates the level of reporter activation by MIB1 overexpression alone. Data are mean + SD (error bar). n = 3.