Fig. 5.

CPNE3 stabilizes YAP1 by competing with β-TRCP for binding to YAP1. A The three-dimensional structure of YAP1 from the Protein Data Bank database. B, C A predictive three-dimensional structural model and model quality evaluation of CPNE3 that was acquired from the Swiss-model database. D The anticipated interface of one of the complexes created when CPNE3 and YAP1 combine. E Immunofluorescence staining for HA-CPNE3 and Flag-YAP1 in BGC823 cells. F, G Results of CPNE3 or YAP1 co-immunoprecipitation (Co-IP) tests carried out in HEK-293 T and BGC-823 cells. H In BGC-823 cells, endogenous Co-IP was performed to evaluate the interaction between CPNE3 and YAP1. I In the cytoplasmic and nuclear protein lysates of BGC-823 cells, the interaction between endogenous CPNE3 and YAP1 was examined independently. J By using glutathione-S-transferase (GST)-pull-down assay, it was shown that His-CPNE3 and GST-YAP1 fusion proteins directly bind to one another. K, L Downregulation of CPNE3 in BGC-823 and MKN-28 cells decreases the expression of YAP1, while proteasome inhibitors MG132 reversed this process. M, N By using the cycloheximide test in BGC-823 cells transfected with siCPNE3-#2 and HGC-27 cells transfected with HA-CPNE3 plasmids, the impact of CPNE3 expression on the stability of YAP1 was evaluated. O Impact of HA-CPNE3 on the ubiquitination of YAP1 was assessed using a BGC-823 cell-based ubiquitination assay. P HEK-293 T cells were transfected with HA-CPNE3, Flag-YAP1, and myc-β-TRCP plasmids, and Co-IP was used to determine the binding status among the three