Fig. 3.

Down-regulation of LATS1 by Itch. (A) Dose-dependent degradation of LATS1 by Itch. Western blot analysis of LATS1 upon expression of increasing amount of Itch in COS7 cells. (B) Loss of Itch ligase activity abolishes its effect on LATS1. Western blot analysis of LATS1-FLAG upon expression of increasing amount of a catalytically inactive Itch (Itch-C830A-Myc) in COS7 cells. (C and D) Loss of interaction of LATS1 and Itch abolishes Itch-induced LATS1 degradation. Western blot analysis of LATS1 upon expression of increasing amounts of Itch-WW-mut (C) or LATS1-Y376A-Y559A upon expressing increasing amounts of Itch (D). (E) Cyclohexmide chase analysis of LATS1 degradation after Itch overexpression. COS7 cells expressing either LATS1-FLAG alone or LATS1-FLAG together with Itch-Myc were treated with cycloheximide (CHX) to inhibit protein synthesis. At an indicated time, cells are harvested and analyzed for LATS1 level using anti-FLAG antibody. (F) Cyclohexmide chase analysis of LATS1 degradation after Itch knockdown. Itch is stably knocked down in MDA-MB231 by shRNA targeting Itch. Cells infected with pGIPZ vector were used as an shRNA control. (G) Western blot analysis of endogenous LATS1 in MCF10A, HeLa, and MCF7 cells upon stable overexpression of Itch. Control, cells without lentiviral infection; WPI, cells stably infected with lentiviral vector WPI; Itch, cells stably infected with lentivirus expressing Itch.