Figure 2.

LRP6 levels and phosphorylation are increased with BVES loss. (A) Cytoplasmic fractionation of 293STF cells following siRNA knockdown of BVES. Cells were treated with 50% Wnt3a conditioned media for 2 h before harvest. pLRP6 quantification is pooled from n = 5 independent experiments, and total LRP6 quantification is pooled from n = 7 independent experiments, each normalized to the control minus Wnt stimulation. (B) LRP6 fold change by qPCR in 2923STF cells with BVES siRNA knockdown. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 8 h before isolation. (C) Cytoplasmic fractionations of 293STF cells with Wnt stimulation time course following BVES knockdown. Cells were treated with 50% Wnt3a-conditioned media for indicated time points. (D) Cytoplasmic fractionations of 293STF or 293 Dishevelled Triple-Knockout (293DVL TKO) cells following BVES knockdown. Cells were stimulated with 50% Wnt3a-conditioned media for 2 h and immunoblotted. β-catenin quantification in the 293DVL TKO is pooled from n = 3 independent experiments. Black line between lanes 6 and 7 is debris on the membrane. (E) Cytoplasmic fractionations of 293STF cells stimulated with 50% control or 50% Wnt3a-conditioned media for 2 h. At the time of Wnt3a stimulation, cells were concurrently treated with dimethyl sulfoxide (DMSO), okadaic acid (OA) at 100 nM or ceramide at 50 μM and probed for pLRP6 (S1490), pGSK3β (S9) and pCREB (S133). Data are representative of two independent experiments. For (A), (B) and (D), *P < 0.05, **P < 0.01, ***P < 0.001 by Mann–Whitney test in the minus Wnt and plus Wnt conditions. ns = non-significant by Mann–Whitney test.