Figure 4

Stabilization of ligand–receptor interaction for the WNT/β-catenin pathway by SPARCL1. (a) Co-IP assay between SPARCL1 and canonical WNT components (receptor: Fzd8 or LRP6; ligand: Wnt3a or Wnt10b). HEK293T cells were transfected with SPARCL1-HA or vector control. The input on the right panel shows the levels of transfected HA-SPARCL1 and endogenous WNT components (Fzd8, LRP6, Wnt3a and Wnt10b) in HA-tagged SPARCL1 or vector control. (b) Brief scheme of the co-culture IP procedure. HEK293T cells were individually transfected with expression plasmids for SPARCL1 or various WNT components (receptor expression plasmid: LRP6 or Fzd8; WNT ligand expression plasmid: Wnt3a or Wnt10b). Eight hours after transfection, cells with receptors and ligands were mixed and co-culture for 24 h, followed by immunoprecipitation assay. (c) SPARCL1 stabilized the binding between canonical WNT protein (Wnt3a or Wnt10b) and Fzd8. HA-tagged Fzd8-expressing cells were co-cultured with myc-tagged WNT ligands-expressing (Wnt3a or Wnt10b) cells and Flag-tagged SPARCL1-expressing cells both separately and in combination. The HA-tagged Fzd8 was immunoprecipitated in this experiment. (d) SPARCL1 stabilized the binding between 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 SPARCL1-expressing cells both separately and in combination. (e and f) Densitometric analysis showed the relative amounts of precipitated WNT ligand (Wnt3a or Wnt10b) interacted with Fzd8 or LRP6 affected by SPARCL1. Values are normalized to intensities without SPARCL1 as 1. (g) Fzd8 CRD co-precipitated LRP6 in the presence of SPARCL1. Flag-tagged Fzd8CRD-expressing cells were co-cultured with myc-tagged SPARCL1-expressing cells and HA-tagged LRP6-expressing cells both separately and in combination. To further confirm the SPARCL1-LRP6-Fzd8 complex model, the Flag-tagged Fzd8CRD was immunoprecipitated in this experiment.