Fig 1: SMOC-2 enhances ligand-receptor interaction of WNT/ß-catenin pathway.(a) Co-IP assay between SMOC-2 and canonical WNT components (ligand: Wnt3a or Wnt10b; receptor: Fzd6 or LRP6). AN3CA cells were transfected with SMOC-2-Flag or a control vector. The input on the right panel shows the levels of transfected Flag-SMOC-2 and endogenous WNT components (Wnt3a, Wnt10b, Fzd6 and LRP6) in Flag-tagged SMOC-2 or vector control.(b) SMOC-2 enhanced the binding between canonical WNT protein (Wnt3a or Wnt10b) and Fzd6. HA-tagged Fzd6-expressing cells were co-cultured with Myc-tagged WNT ligand-expressing (Wnt3a or Wnt10b) cells and Flag-tagged SMOC-2 expressing cells both separately and in combination. The HA-tagged Fzd6 was immunoprecipitated in this experiment.(c and d) Densitometric analysis showed the relative amounts of precipitated WNT ligand (Wnt3a or Wnt10b) interacted with Fzd6 or LRP6 affected by SMOC-2. Values are normalized to intensities without SMOC-2 as 1.(e) SMOC-2 enhanced the binding between the canonical WNT protein (Wnt3a or Wnt10b) and LRP6. Similarly, HA-tagged LRP6-expressing cells were co-cultured with WNT ligands-expressing cells and Flag-tagged SMOC-2-expressing cells both separately and in combination.(f) The interactions of SMOC-2 and LRP6/Fzd6 were detected by in situ proximity ligation assay (red dots, n = 3, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Experiments were statistically analyzed using two-tailed Student's t-test). Scale bars: 20 µm.(g) Vector, SMOC-2, SMOC-2 + siLRP6 and SMOC-2 + siFzd6 groups of HEC-1A and ECC-1 cells were treated with a series of concentrations paclitaxel/cisplatin to obtain half maximal inhibitory concentration (IC50). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)