Fig. 3

Usp7 Counteracts Ci Degradation Mediated by Both Slimb-Cul1 and Hib-Cul3 E3 Ligases UAS transgene expression with MS1096 gal4 is usually stronger in the dorsal region than in the ventral region of wing pouch. (A) A wing disc of MS1096 line was stained to show full-length Ci (green). (B and B′) A wing disc expressing Fg-usp7 with MS1096 was immunostained with Fg (white) and Ci (green) antibodies. Ci was accumulated when usp7 was overexpressed (arrow). (C–C′′) A wing disc expressing Fg-usp7 with apG4 was immunostained to show the expression of GFP (green) and Ci (white). (D and D′) Usp7-HA (green) expressed in S2 cells mainly localized in nuclei. (E and E′) Fg-Usp7 protein (green) expressed by sgs3-gal4 in salivary glands mainly localized in nuclei. (F and F′) Fg-Usp7 protein (green) expressed by MS1096 in wing discs mainly localized in nuclei. From (D) to (F′), the nuclei were marked by DAPI (blue). (G–I′′) A MS1096 wing disc (G) and the discs expressing Fg-slimb alone (H–H′) or Fg-slimb/HA-usp7 together (I–I′′) by MS1096 were immunostained to show Fg tag (green), Ci (white), and HA tag (red). Usp7 could counteract Ci degradation by Slimb-Cul1 E3 ligase (arrows). (J–L′′) A MS1096 wing disc (J) and the discs expressing HA-hib alone (K–K′) or HA-hib/Fg-usp7 (L–L′′) together by MS1096 were immunostained with HA tag (green), Ci (white), and Fg tag (red) antibodies. Usp7 could attenuate Ci degradation by Hib-Cul3 E3 ligase (arrows). (M and N) Western blot analysis of lysates from control wing discs or wing discs expressing indicated RNAi using MS1096 driver. Approximately 40 discs were dissected, lysed, and blotted with rabbit anti-CiN antibody. Signal ratio of CiR to CiFL of each lane was shown on the right. The result is presented as means ± SD of values from three independent experiments. See also Figure S4.