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. 2017 Apr 17;114(18):4691–4696. doi: 10.1073/pnas.1620306114

Fig. 2.

Fig. 2.

YOD1 regulates LATS1 stability in an ITCH-dependent manner. (A) Assessment of levels of major Hippo components in HEK293T cells transfected with siRNAs for GFP or YOD1. (B) HEK293T cells were transfected with increasing amounts of YOD1 and analyzed via Western blotting for the indicated proteins. (C) (Left) HEK293T cells were cotransfected with the indicated siRNAs, pRL-TK, and 8xGTIIC luciferase. (Right) A representative Western blot indicating the successful knockdown of proteins by siRNAs. (D) Protein lysates from HEK293T cells were immunoprecipitated with an anti-ITCH antibody and analyzed via immunoblotting for the indicated proteins. WCL, whole-cell lysates. (E) Empty vectors, Flag-YOD1-WT, or Flag-YOD1-CS were expressed in HEK293T cells followed by Western blotting with the indicated antibodies. (F) HEK293T cells were transfected with a scrambled siRNA or three different YOD1 siRNAs targeting different regions. One day after siRNA transfection, the cells were harvested and analyzed via Western blotting for the indicated proteins. (G) HEK293T cells were transfected with either siGFP or siITCH. One day after siRNA transfection, the cells were cotransfected with the indicated plasmids and reporter constructs. Reporter activity was measured 24 h after the latter transfection. (H) HEK293T cells transfected with either siScrambled or siYOD1 were treated with either DMSO (control) or a proteasome inhibitor (MG132). (I) HA-ubiquitin was cotransfected with empty vectors, Flag-YOD1-WT, or Flag-YOD1-CS into HEK293T cells, which were treated with 25 µM MG132 for 4 h before being harvested. Total cell lysates were immunoprecipitated with an anti-ITCH antibody, followed by immunoblotting with an anti-HA antibody. (J) The experiments were performed as described in I except that siRNA targeting YOD1 was used. (K) HEK293T cells were transfected with the indicated plasmids and siRNAs and were treated with 25 µM MG132 for 4 h before being harvested. Total cell lysates were immunoprecipitated with an anti-LATS1 antibody, followed by immunoblotting with an anti-HA antibody. The statistical results represent average values from a representative experiment performed in triplicate. Error bars indicate SDs of triplicate assays. **P < 0.01. CS, C160S (YOD1 mutant); IP, immunoprecipitation.