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. 2015 Sep 30;6(34):36019–36031. doi: 10.18632/oncotarget.5935

Figure 5. Mitotic phosphorylation is required for YAP's transcriptional activity.

Figure 5

A., Luciferase reporter assays in HEK293T cells. Cells were transfected with reporters and various YAP constructs and harvested 48 hours post-transfection for measuring luciferase activity. Expression levels of YAP (and mutants) are similar (data not shown). Ctrl: control (empty vector); YAP3A: T119A/S289A/S367A; 4A: S127A/3A. N.S.: not significant. Data are expressed as the mean ± s.e.m. of three independent experiments. ***: p < 0.001(t-test). B., Mitotic phosphorylation impacted YAP's binding to Lats2. HEK293T cell were transfected with various DNA constructs as indicated and were harvested 48 hours post-transfection for immunoprecipitation (IP). C., Mitotic phosphorylation did not significantly affect YAP's binding to TEAD1. HEK293T cell were transfected with various DNA constructs as indicated and were harvested 48 hours post-transfection for IP. D., HeLa cells were synchronized by a double thymidine (DT) block and release method. Endogenous YAP was immunoprecipitated at the indicated time points and probed with the indicated antibodies. Total cell lysates before IP were also analyzed to confirm the cell phase status. E., HPNE cells expressing vector, YAP, YAP-S127A, YAP-S127A/3A (YAP4A) or YAP3D were probed with the indicated antibodies. F., Quantitative RT-PCR of known YAP targets in HPNE cells. Data are expressed as the mean ± s.e.m. of three independent experiments. **: p < 0.01; *: p < 0.05 when compared to vector control (t-test).