Fig. 6: USP12 interacts with YAP proteins and regulates YAP protein stability via the proteasome pathway.

A Immunofluorescence staining assay showing the localization patterns of USP12 and YAP in AGS cells. Intracellular localization of USP12 (green) and YAP (red) is shown. Nucleus (blue) were stained with DAPI. Scale bar, 20 µm. B, C Co-IP showing the endogenous interaction between USP12 and YAP. Llysates of AGS cells were precipitated with anti-YAP or anti-USP12 antibodies, and the precipitates were examined by immunoblotting. D Immunoblot analysis showing the expression level of YAP protein in siControl and siUSP12 expressing AGS cells by treat with 10 μM proteasome inhibitor MG132. E, F Immunoblot analysis showing USP12 deletion decreased YAP half-life. siControl and siUSP12 were transfected in AGS cells. Cells were treated with 100 μM cycloheximide (CHX) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control (E). The expression of YAP protein was estimated by ImageJ software and is represented graphically (F). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. G, H USP12 deubiquitinating enzyme inactivity mutant cannot increase YAP half-life. AGS cells were transfected with 1 μg Flag or Flag-USP12^WT/Flag-USP12^C48S for 24 h. Then cells were treated with 100 μM cycloheximide (CHX) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control (G). The expression of YAP protein was estimated by ImageJ software and is represented graphically in the right panel (H). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.