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. 2021 Jan 8;11:40. doi: 10.1038/s41598-020-79524-3

Figure 2.

Figure 2

Mapping of the GPC3 binding region on FAT1. (A) Diagram of the FAT1 fragments, either with C-terminal FLAG tag for co-IP assays, or with C-terminal hFc tag for ELISA and FACS experiments. (B) Co-IP of GPC3 by FAT1 truncation fragments with C-terminal FLAG tag in 293 T cells. GPC3 and FAT1 truncation fragments were co-expressed in 293 T cells. FAT1 fragments were pulled down with anti-FLAG monoclonal antibody, and the co-immunoprecipitated GPC3 was probed with anti-GPC3 monoclonal antibody hYP7. Ctrl was an empty expression vector for FAT1 fragments. Full-length blots/gels were presented in Supplementary Figure S2. (C) ELISA analysis of recombinant FAT1 fragments binding to immobilized GPC3. GPC3-His was coated on the 96-well plate (5 μg/ml, 50 μl/well), and varied amount of FAT1 fragments was added to the wells. Pooled hIgG was used as isotype control. (D) Flow cytometry analysis of the binding specificity of the recombinant FAT1 fragments to cell surface GPC3. A pair of GPC3 negative A431 cell line and GPC3 over-expressing A431 (GPC3+) cells were tested by incubation with hFc-tagged FAT1 fragments (50 μg/ml). Blue solid line, FAT1 fragments staining on A431 (GPC3+) cells; Blue dashed line, FAT1 fragments staining on A431; red line, isotype control staining on A431 (GPC3+); Shaded area, isotype control staining on A431. (E) Binding specificity of recombinant FAT1 fragments to cell surface GPC3 that was calculated by the ratio of A431 (GPC3+) binding (geometrical mean of fluorescence intensity) versus A431, based on data from (D). Data represent mean ± SEM (n = 3). (F) Schematic diagram of the GPC3 binding region on FAT1 based on the above data.