Fig. 6

The regulation of Usp7 on Hippo (Hpo) pathway is conserved. a, b Wing discs expressing Fg-hausp by En-gal4 were stained to show Fg (red) and ban-lacZ (white in a) or diap1-lacZ (white in b). All crosses were set at 29 °C. c, d Overexpression of hausp could restore the decrease of ban-lacZ (c) and diap1-lacZ (d) caused by usp7 knockdown. Scale bars: 50 μm for all images. e Herpes virus-associated ubiquitin-specific protease (HAUSP) bound Yorkie (Yki) in S2 cells. The arrowhead indicates IgG. f HAUSP interacted with Yap. g, h Endogenous Yap reciprocally pulled down endogenous HAUSP in 293T cells. The cells from one 10 cm plate were lysed and the lysis was equivalently divided two parts for IP with control IgG or Yap/HAUSP antibodies. The expression of corresponding proteins from whole-cell lysis (WCE) was shown below. i LATS1/LATS2 decreased the interaction between HAUSP and Yap. j MST1/MST2 decreased the interaction between HAUSP and Yap. k Transfection of Fg-HAUSP increased endogenous Yap protein level in 293T cells. Actin acts as a loading control. l Knockdown of endogenous hausp decreased Yap protein, whereas did not affect TEAD level. Actin acts as a loading control. m Immunoblots of lysates from 293T cells transfected with indicated constructs. Notably, β-TrCP destabilized Yap in a dose-dependent manner, which was hampered by HAUSP, not HAUSP-CA. Actin acts as a loading control. n Immunoblots of immunoprecipitates (top) or lysates (bottom three panels) from 293T cells expressing indicated proteins and treated with MG132 plus NH₄Cl for 4 h. HAUSP decreased, whereas HAUSP-CA increased the ubiquitination of Yap protein. o, p HAUSP decreased, whereas HAUSP-CA increased β-TrCP-mediated Yap ubiquitination. q Relative mRNA levels of indicated genes from 293T cells with or without hausp knockdown were revealed by real-time PCR. r Relative mRNA levels of indicated genes from 293T cells with or without HAUSP transfection were revealed by real-time PCR. s CTGF-luciferase reporter assay in 293T cells transfected with indicated constructs or siRNAs. CTGF-luciferase activities were normalized to renilla luciferase activities. For statistical results, data are means ± SEM. n = 3 biological-independent experiments. In all above results, ^**P < 0.01, ^***P < 0.001, ns not significant, by Student’s t-test