FIGURE 6.

PKN1 co-purifies with the WNT3A receptor FZD7, and its depletion promotes phosphorylation changes associated with Wnt/β-catenin signaling. A, proteins (nodes) are represented as circles (bait) or squares (prey) with the relationships between nodes (edges) denoted by the connecting lines. A protein-protein interaction network for FZD7 and PKN1 was generated from primary affinity purification-mass spectrometry data (blue edges) and from literature-curated interactions (red edges). Red nodes represent bait or prey that has been previously reported to regulate Wnt/β-catenin signaling. B, HA or the negative control CCND1 was immunoprecipitated (IP) from lysates isolated from HEK293T cells stably expressing HA-FZD7 and analyzed by Western blot (WB). C and D, A375 malignant melanoma cells were transiently transfected with the indicated siRNA oligonucleotides. 62 h after transfection, the cells were stimulated for the indicated times and analyzed by Western blot with the indicated antibodies. D, Western blots from three independent experiments were quantified by densitometry for phosphorylated CTNNB1 (P(S33)CTNNB1; left panel), total LRP6 (LRP6; middle panel), and phosphorylated LRP6 (p-LRP6; right panel). Error bars represent S.E., and p values (*, p < 0.05; **, p < 0.01; ****, p < 0.0001) were calculated using two-way ANOVA (right brackets) followed by a Bonferroni post-test (individual time points). CTRL, control.