LATS and AMOT proteins are required for DUB3-mediated regulation of Hippo signaling.

(A) Immunoblots showing the effect of DUB3 expression on LATS kinases, AMOT and YAP. HEK293T cells were transfected with a vector expressing Flag-DUB3 or a control vector together with a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2 or a scrambled siRNA control. Blots were probed with antibodies against Flag, AMOT, AMOTL1, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. Samples were run on the same SDS-acrylamide gels with intervening lanes removed. (B) Immunoblots showing the effect of DUB3 depletion on LATS kinases, AMOT and YAP. HEK293T cells were transfected with a vector expressing Flag-DUB3 or a control vector together with a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2 or a scrambled siRNA control. Blots were probed with antibodies against DUB3, AMOT, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. Samples were run on the same gels with intervening lanes removed. (C) Immunoblots showing the effect of DUB3 depletion on ITCH, LATS kinases and AMOT. HEK293T cells were transfected with a control or DUB3 siRNA in the presence of LATS1 and LATS2 siRNAs or a scrambled siRNA control. Blots were probed with antibodies against DUB3, ITCH, AMOT, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. (D) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or DUB3 siRNA in the presence of a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2, a mixture of siRNAs targeting LATS1 and LATS2 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance).