Fig. 6: Engineered GPC3 interacts with FZD-7.

Cells transfected with GPC3-O and FZD-7 were incubated with alkyneHaHS and labeled with an azide group by metabolic labeling. The engineered GPC3 and FZD-7 were linked by click reaction. For the binding assay, cell lysates were added to wells covered with a GPC3 antibody, and the bound FZD-7 was detected using an anti-FZD-7 antibody. For the WB assay, protein samples were run through 8% SDS-PAGE and the transferred membranes were incubated with GPC3 or FZD-7 antibody and the corresponding secondary antibody. a Schematic of the experiment performed to detect the interaction between GPC3 and FZD-7. b Binding assay between GPC3 and FZD-7. Bars represent the absorbance at 405 nm (mean ± SD of triplicates). c Protein interactions analyzed by WB using FZD-7 antibody. Lane 1, cells were transfected with FZD-7 and wild-type GPC3, and then metabolically labeled with GlcNAz; Lane 2, cells were transfected with FZD-7 and engineered GPC3-O, metabolically labeled with GlcNAz, and then alkyneHaHS DP14 as side chains were attached to the GPC3-O on the cell surface; Lane 3, cells were transfected with FZD-7 and engineered GPC3-O, metabolically labeled with GlcNAz, incubated with HaHS DP14 without alkyne group, and then the click chemical reaction was performed; Lane 4, cells were transfected and metabolically labeled as in lane 2 and the click chemical reaction was performed; Lane 5, cell lysates prepared as in lane 3 were treated with heparinases before the click reaction; Lane 6, GPC3 and conjugated proteins in cell lysates generated as in lane 3 were purified with an affinity column with immobilized anti-GPC3 antibody. d Protein interactions analyzed by WB using GPC3 antibody. Lane 1, cells were transfected with FZD-7 and wild-type GPC3, and then metabolically labeled with GlcNAz; Lane 2, cells were transfected with FZD-7 and engineered GPC3-O, metabolically labeled with GlcNAz, and then alkyneHaHS DP14 side chains were attached to the GPC3-O on the cell surface; Lane 3, cells were transfected and metabolically labeled as in lane 2, and a click chemical reaction was performed; Lane 4, cell lysates prepared as in lane 3 were treated with heparinases before the click reaction; Lane 5, GPC3 and its conjugated proteins in cell lysates generated as in lane 3 were purified with an affinity column with immobilized with anti-GPC3 antibody; G3, core protein of GPC3-O; G3-2, GPC3-O with two HS DP14 chains; G3-1, GPC3-O with one HS DP14 chain; F7, FZD-7; G3-2&F7, the complex of GPC3-O with two HS DP14 chains and FZD-7; G3-1&F7, the complex of GPC3-O with one HS DP14 chains and FZD-7. All experiments were repeated three times with similar results. Source data are provided in a Source Data file.