Fig. 4: Specific binding of E8 nanobody to YAP.

A Ectopic expression of YAP in MKN45 YAP-null cells was confirmed by western blot. MKN45 cells stably expressing either an empty vector (MKN45-vec) or YAP (MKN45-YAP) were generated by lentiviral infection using pLVX-empty vector or pLVX-YAP constructs. B Western blot analysis of E8 nanobody binding using cell lysates from MKN45-vec or MKN45-YAP. C Dot blot assay examining the binding of E8 to cell lysates from MKN45-vec or MKN45-YAP. D In-Cell ELISA analysis showing the binding of E8 nanobody to MKN45-YAP cells but not MKN45-vec cells (n = 3 biological replicates, mean ± SEM). E Immunofluorescence analysis showing specific recognition of E8 nanobody to YAP protein in MKN45-YAP cells, but not in MKN45-vec cells. Scale bar: 20 μm. F ELISA analysis evaluating the binding ability of E8 to recombinant YAP fragment proteins (n = 2 biological replicates, mean ± SEM). GST served as a negative control. Data are representative of two independent experiments. G Co-immunoprecipiatation (coIP) demonstrating the interaction between Flag-YAP (155-504 aa) and HA-E8. Flag-YAP was cotransfected with HA-C3 (irrelevant control nanobody) or HA-E8 in HEK293T cells. Immunoprecipitated HA-tagged proteins were analyzed for co-precipitation of Flag-YAP by Western blot. H Schematic diagram of YAP deletion constructs (A1/2/3/4 and B1/2/3). TB, TEAD-binding domain; TAD, C-terminal transactivation domain. I The YAP region spanning 155–290 aa is required for its interaction with E8. YAP deletion constructs A1/2/3/4 were cotransfected with HA-E8 into HEK293T cells, and coIP was performed to identify interacting regions. J WW2 domain of YAP mediates its interaction with E8. CoIP experiments, similar to (I), were performed using YAP deletion mutants B1/2/3. Experiments in figures (A–C, E, G, I, J) were repeated twice or more. Source data are provided as a Source Data file.