Fig. 4

Usp7 Attenuates Slimb-Cul1- and Hib-Cul3-Mediated Ci Ubiquitination through Its Deubiquitinating Activity (A) Both Fg-Usp7 and Fg-Usp7CA could bind Myc-Ci in S2 cells. (B–E) Comparison of adult wing phenotypes from control flies (B), usp7 knockdown (C), Fg-usp7 expression in usp7 knockdown background (D), and Fg-usp7CA expression in usp7 knockdown background (E). Arrows mark the space between vein 3 and vein 4. (F–F′′) A wing disc expressing usp7CA with MS1096 was immunostained to show Fg tag (white) and Ci (green). Ci level was not upregulated by usp7CA expression (arrow). (G–G′′) A wing disc expressing usp7CA with MS1096 was stained for Fg tag (white) and dpp-lacZ (green). usp7CA overexpression decreased dpp-lacZ expression (arrow). (H and H′) A wing disc expressing usp7-ΔMATH with MS1096 was stained with Fg tag (white) and Ci (green) antibodies. Ci level was not altered by usp7-ΔMATH expression (arrow). (I–L) Wing discs expressing usp7 RNAi (I), Fg-usp7/usp7 RNAi (J), Fg-usp7CA/usp7 RNAi (K), or Fg-usp7-ΔMATH/usp7 RNAi (L) were stained for dpp-lacZ expression (white). The attenuation of dpp-lacZ caused by usp7 knockdown (I, arrow) could be restored by the expression of Fg-usp7 (J, arrow), but not Fg-usp7CA (K, arrow) and Fg-usp7-ΔMATH (L, arrow). (M) Western blots of immunoprecipitates (top) or lysates (bottom three images) from S2 cells expressing indicated proteins and treated with MG132. (N) Knockdown of usp7 using usp7-dsRNA promoted Ci ubiquitination in S2 cells. (O) usp7-dsRNA could effectively knock down Fg-usp7 expression in S2 cells. Actin acts as a loading control. (P and Q) Transfected S2 cells were treated with MG132 for 4 hr before cell harvesting. Fg-Usp7 decreased, but Fg-Usp7CA promoted Ci ubiquitination mediated by Slimb-Cul1 (P) and Hib-Cul3 (Q). From (M) to (Q), quantification analyses of the ubiquitination levels of Ci are shown below each autoradiogram. Data are means ± SD from three independent experiments.