Fig. 2
SKP2 interacts with YAP and induces its K63-linked polyubiquitination. a The HEK293T SFB-YAP stable cell line was transfected with MYC-tagged E3 ligases, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against FLAG and MYC. b The HEK293T SFB-YAP stable cell line was co-transfected with the K63-specific mutant of HA-ubiquitin and the indicated E3 ligase, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. c Co-immunoprecipitation of endogenous SKP2 with endogenous YAP. d In vitro binding of bacterially purified MBP-SKP2 to in vitro translated and purified GST-YAP. e In vitro binding of bacterially purified MBP-SKP2 to SFB-YAP purified from the HEK293T SFB-YAP stable cell line. MBP-SKP2 or MBP protein was incubated with SFB-YAP protein with or without CIP treatment at 37 °C for 1 h, followed by immunoblotting with antibodies against SKP2, p-YAP (S127), and FLAG. f The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48R or K63R) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. g The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48-specific or K63-specific) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. h Total K63-linkage specific ubiquitinated proteins in control or SKP2 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin. i siRNA targeting CUL1 or SKP1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours after siRNA transfection, cells were transfected with HA-ubiquitin and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j The HEK293T UBCH5c shRNA stable cell line was transfected with SFB-YAP and HA-ubiquitin with or without MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. k Purified GST-YAP protein was incubated with ATP, E1, UBCH5c, and the K63-specific mutant of His-ubiquitin with or without the SCFSKP2 complex, followed by immunoprecipitation with a YAP-specific antibody and immunoblotting with antibodies against ubiquitin and YAP
