Fig. 5.

Usp7 deubiquitinates and stabilizes Yorkie (Yki). a Immunoblots of lysates from S2 cells transfected with Myc-Yki and treated by CHX for indicated intervals. To prevent the function of lysosome or proteasome, the cells were treated with corresponding inhibitors. Of note, Myc-Yki was unstable, whereas lysosome inhibitor could hamper its degradation. Actin acts as a loading control. Quantification analyses were shown on right. b Endogenous Yki protein was unstable and underwent lysosome-mediated degradation. Actin acts as a loading control. Quantification analyses were shown on right. c S2 cells treated by CHX for indicated intervals were analyzed by subcellular fractionation. Of note, the cytoplasmic Yki was degraded by lysosome, whereas the nuclear Yki was degraded by proteasome. Lamin C and Tubulin are used as loading controls. d Myc-Yki-NLS protein was degraded by proteasome in S2 cells. Actin acts as a loading control. Quantification analyses were shown on right. e Myc-Myr-Yki protein was degraded by lysosome in S2 cells. Actin acts as a loading control. Quantification analyses were shown on right. f Immunoblots of lysates from S2 cells expressing indicated constructs and treated with CHX for indicated intervals. Quantification analyses were shown on right. Notably, Usp7 effectively blocked Yki degradation. g Knockdown of usp7 promoted Yki degradation. h S2 cells transfected with indicated constructs were analyzed by subcellular fractionation. Of note, Usp7 increased the nuclear Yki protein levels. Quantification analyses were shown on right. Lamin C and Tubulin are used as loading controls. i Usp7 increased Myc-Yki-NLS protein level. j Knockdown of usp7 decreased nuclear Yki protein. k Immunoblots of immunoprecipitates (top) or lysates (bottom three panels) from S2 cells expressing indicated proteins and treated with MG132 plus NH₄Cl for 4 h. Knockdown of usp7 promoted Yki ubiquitination. l Usp7 and Usp7-ΔMATH attenuated, but Usp7-CA promoted endogenous Yki ubiquitination. m Usp7 attenuated, whereas Usp7-CA promoted Yki-NLS ubiquitination. For statistical results, data are means ± SEM. n = 3 biological-independent experiments. Above all, the arrowhead indicates heavy IgG