Cells were washed in PBS, dissolved in 60‐100 μL solubilizing solution (9M urea, 2% NP40), and subjected to immunoblotting as previously described.3 (link) Primary Abs recognizing the following proteins were used: YAP1 (D8H1X), TAZ (v386), phospho‐(p)‐YAP1 (Ser127), p‐YAP1 (Ser397), p‐TAZ (S89), LATS1, and p‐LATS1 (Ser909) (all from Cell Signaling Technology), actin (Sigma), or GAPDH (Wako Chemical). Horseradish peroxidase‐conjugated anti‐rabbit Ab (Cell Signaling Technology) was used as the secondary Ab. Band intensities were determined by Fujifilm Multi Gauge software or the ImageJ program. The YAP1 and TAZ protein levels were normalized to actin or GAPDH, as indicated. Phosphorylated YAP1, p‐TAZ, and p‐LATS1 were normalized to total YAP1, TAZ, and LATS1 levels, respectively.
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Immunoblotting Analysis of Hippo Pathway
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Corresponding Organization : Kyushu University
Other organizations : Fujifilm (Japan), University of Occupational and Environmental Health Japan, Tokyo Medical and Dental University, University of Toronto, Chinese University of Hong Kong, University of Hong Kong, Princess Margaret Cancer Centre, University Health Network, RIKEN Center for Biosystems Dynamics Research, RIKEN Center for Sustainable Resource Science, The University of Tokyo
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Variable analysis
independent variables
- Solubilizing solution (9M urea, 2% NP40)
- YAP1 protein levels
- TAZ protein levels
- Phosphorylated YAP1 (Ser127, Ser397)
- Phosphorylated TAZ (S89)
- LATS1 protein levels
- Phosphorylated LATS1 (Ser909)
- Actin
- GAPDH
- Positive controls: Not explicitly mentioned.
- Negative controls: Not explicitly mentioned.
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