Fig. 9

Xiap may act as an E3 ligase to catalyze Yap1 neddylation. A, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with plasmids expressing HA-yap1 and PCI-His empty vector or His-nedd8 for 24 h. B, neddylation of yap1 is abolished by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with plasmids expressing Myc-yap1 and a conjugable mutant nedd8 (His-nedd8-?GG) for 24 h. C, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with Myc-yap1, His-nedd8, and treated with MLN4924 (1 ?M) for 12 h (third lane) or transfected with Myc-yap1, His-nedd8, HA-uba3, and HA-ubc12 (fourth lane). D, protein lysates from zebrafish brains were immunoprecipitated with mouse lgG (control) or anti-YAP1 antibody, respectively. Under partially denaturing conditions, coimmunoprecipitation was detected with anti-Nedd8 antibody. E, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with HA-nedd4a, HA-xiap, HA-smurf1, and HA-mdm2 for 24 h. F, the interaction between yap1 and xiap is detected by immunoprecipitation with anti-FLAG agarose and Western blot in HEK293T cells transfected with the indicated plasmids for 24 h. G, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with HA-xiap or xiap mutant (HA-xiap-H374A) for 24 h. IB, immunoblotting; IP, immunoprecipitation; TCL, total cell lysate; YAP1, yes-associated protein 1.