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. 2016 Dec 15;18(1):72–86. doi: 10.15252/embr.201642681

Figure EV1. Osmotic stress induces YAP phosphorylation but not cytoplasmic retention.

Figure EV1

  1. Osmotic stress‐induced YAP phosphorylation is dose‐dependent. HEK293A cells were treated with different doses of sorbitol for 30 and 60 min. YAP phosphorylation is determined by mobility shift on phos‐tag gel and a S127 phosphospecific antibody.
  2. Sorbitol stimulates YAP phosphorylation in MCF10A cells. MCF10A cells were cultured in the presence or absence of serum, and were treated with 0.2 M sorbitol for the indicated time points. A phos‐tag gel was used to assess total YAP phosphorylation.
  3. NaCl stimulates YAP and LATS phosphorylation. HEK293A cells were cultured in the presence of serum and were treated with 0.1 M NaCl for the indicated time points. A phos‐tag gel was used to assess YAP phosphorylation.
  4. NaCl does not induce YAP cytoplasmic localization. HEK293A cells were treated with 0.1 M NaCl for 1 h in the presence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining with an antibody that recognizes both YAP and TAZ (red). DAPI (blue) was used to stain for DNA (cell nuclei). Scale bars: 20 μm. Quantification of YAP/TAZ more nuclear (N > C) or more cytosolic (N < C) is determined with randomly chosen fields, each with approximately 100 cells.
  5. Sustained osmotic stress induces YAP cytoplasmic localization in the presence of serum. HEK293A cells were treated with 0.2 M sorbitol from 30 min to 8 h in the presence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bars: 20 μm.

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