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. 2019 Feb 25;20(4):e46570. doi: 10.15252/embr.201846570

Figure 3. UBTD1 promotes YAP degradation by the UPS independently of the Hippo signaling pathway.

Figure 3

  • A
    Immunoblot of DU145 and A549 cells at various cell densities ranging from 20 to 100% confluence showing the increased expression of UBTD1, p‐YAP (ser127), and YAP levels with the cell confluency. Tubulin was used as a loading control.
  • B–E
    DU145 cells or (F) A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p‐YAP (ser127), YAP, and UBTD1 (left) and quantification of YAP and p‐YAP/YAP levels (right) in control or UBTD1‐depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA‐mediated depletion. Actin was used as a loading control. (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p‐ser909LATS and p‐thr183MST1/p‐thr180MST2) and total LATS, and MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA‐mediated depletion. Actin and RhoGDI were used as loading controls. In (B) and (C), immunoblots for the loading control (actin) and UBTD1 knock‐down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels. (D, E) Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 or (E) A549 UBTD1‐depleted cells treated or not with cycloheximide (CHX) for 2 h. Immunoblot of UBTD1 shows the level of siRNA‐mediated depletion. Actin was used as a loading control.
  • F
    Immunoblots showing YAP levels in UBTD1‐depleted confluent DU145 (left) or A549 (right) cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA‐mediated depletion. Actin was used as a loading control. Numbers at the bottom of the Western blots represent the ratio between 6h treatment/0h for each condition.
Data information: n ≥ 3 independent experiments; *P < 0.05; (B) Bonferroni's multiple comparison test; data are mean ± s.e.m.Source data are available online for this figure.