Fig. 4.

Decreased transcriptional activity via disruption of YAP’s leucine zipper motif is not dependent on insert length. (A) Schematic illustration of the FLAG-tagged GAL4-YAP TAD fusion constructs. The transactivation domain (blue) and PDZ-binding motif (black) for hYAP isoforms (α, β, γ and δ) and mYAP, were cloned 3′ to the FLAG sequence using unique BamHI/XbaI sites. HEK293T (B), D645 (C) and HeLa (D) cells were transfected with pFR-Luc (Con), together with FLAG-tagged empty vector (EV), or the C-terminal region of Stat5a, VP16, mYAP (YAP^m), or the four hYAP isoforms (YAP^α, YAP^β, YAP^γ or YAP^δ) fused to the GAL4 DNA-binding domain. After 24 h cells were harvested and luciferase activity was determined. Firefly luciferase activity was normalised to Renilla luciferase activity. The average luciferase activities were then normalised to YAP^α, which was set to 100%. Data is presented as mean+SEM from three (B) or four (C and D) independent experiments. *p<0.05, **p<0.01, ***p<0.001. Lower panel (B): Cell lysates were separated by SDS-PAGE, transferred to membrane and immunoblotted for FLAG and β-Actin as indicated. Size markers are shown in kilodaltons.