Fig. 7
SKP2 and K63-linked ubiquitination enhance the YAP-TEAD interaction. a HEK293A cells were stably transfected with SFB-YAP and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. H: high density; L: low density. b The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2 and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. c The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2. SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. d HEK293T cells were transfected with SFB-YAP (WT or the 2KR mutant). SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. e SKP2 forms a complex with YAP and TEAD1. HA-TEAD1 and MYC-SKP2 were co-transfected with or without SFB-YAP into HEK293T cells. SFB-YAP was pulled down with streptavidin beads and then eluted with biotin buffer. The eluded proteins were subjected to immunoprecipitation with a MYC-specific antibody to pull down MYC-SKP2 and immunoblotting with antibodies against FLAG, HA, and MYC
