Fig. 6

HAUSP Binds and Deubiquitinates Gli Proteins (A) Adult wing that expresses usp7 RNAi and Fg-tagged hausp. Arrow marks the space between vein 3 and vein 4. (B) Fg-HAUSP could bind Myc-Ci in S2 cells. (C and C′) A wing disc of apG4 was stained to show GFP (green) and Ci (white). (D and D′) Overexpression of hausp by apG4 resulted in an increase of Ci (white, arrow) in wing discs. (E–G) Fg-HAUSP could bind Myc-Gli1, Myc-Gli2, and Myc-Gli3 in 293T cells, respectively. The expression of corresponding proteins from cell lysate (CL) is shown below. (H) Endogenous HAUSP pulled down endogenous Gli1, Gli2, and Gli3 in 293T cells. The cells from one 10-cm plate were lysed and the lysate was equally divided into two parts for IP with control IgG or HAUSP antibody. (I) ShhN treatment increased the interaction between endogenous HAUSP and endogenous Gli proteins in 293T cells. (J and K) Sufu expression inhibited the interaction between HAUSP and Myc-tagged Gli1 (J) and Gli2 (K) in Sufu−/− MEF cells. The transfected cells were treated with MG132 for 4 hr prior to cell harvesting. (L–N) HAUSP blocked Gli protein degradation mediated by corresponding E3 ligases. 293T cells were transfected with indicated plasmids. Actin is shown as a loading control. RT-PCR analysis was performed to determine the levels of Myc-gli mRNA. The forward primer was on Myc tag, whereas the reverse primer was on gli. actin acts as a control. (O–Q) HAUSP inhibited, but HAUSPCA promoted the ubiquitination of Gli proteins mediated by E3 ligases. 293T were transfected with indicated plasmids and treated with MG132 for 4 hr before cell harvesting. Quantification analyses of the ubiquitination levels of Gli proteins are shown on right. The results were presented as means ± SD of values from three independent experiments. See also Figure S5.