Fig. 4.

DVL has nuclear export function on phosphorylated YAP. a Relative fold repression of reporter activities and nuclear YAP abundance by DVL on wt or phospho-resistant mutants of YAP (S127A, 5SA) were measured with TEAD reporter assay (left panel) and immunoblot analysis (right panels), respectively. Relative nuclear YAP abundance compared to control was measured by ImageJ. b The MCF-7 cells were transfected with HA-tagged DVL3 in combination with flag-tagged wt or phospho-resistant mutants YAP (S127A, 5SA), and nuclear localizations of YAP and DVL were examined by confocal immunofluorescence microscopy. To minimize the overexpression issue, 10 ng of YAP expression vectors was used. Scale bar, 10 μm. c YAP and DVL3 were co-transfected in combination with vector control or dominant negative Lats2 (Lats2-KR), and relative TEAD reporter activity was measured. Data presented as mean ± SD, n = 3. d DVL3 was transfected in wt or Lats1/2 double-knockout (Lats1/2^−/−) 293A cells, and nuclear YAP abundance by DVL3 was determined by immunoblot analysis (left panels) and immunofluorescence study (right panels). The cells were serum-starved for 16 h before harvest. e Schematic diagram of nuclear export of phosphorylated YAP by DVL. f Wnt ligands activate YAP resulting in decreased interaction with DVL. The 293A cells transfected with HA-tagged DVL3 were serum-starved and then stimulated by Wnt1 and Wnt3a ligands for 4 h. The whole-cell lysates (WCL) were subjected for immunoblot analysis and immunoprecipitation (IP) assay with anti-HA antibody. g Soluble Wnt1 and Wnt3a induce YAP nuclear localization. The confluent 293A cells were serum-starved and then stimulated by Wnt ligands for 4 h. Endogenous YAP and DVL localization were determined by confocal immunofluorescence microscopy. Scale bar, 10 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 10