Figure 5A. Knockout of NLK reduces the interaction between endogenous YAP and TEAD. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-TEAD1 antibody and immunoblotted with anti-TEAD1 antibody or anti-YAP antibody (top panel). Whole cell lysates (WCL) were immunoblotted with antibodies indicated in the figure.B. Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies in the figure.C. Downregulation of nemo with RNAi achieved in the posterior compartment of wing imaginal discs (co-expression of GFP serves to mark the posterior compartment) using the hh-Gal4 driver results in decreased expression of the Yorkie target gene DIAP1. Two different RNAi constructs produce similar effect. Panels below show Z-stacks of magnified areas around the A/P border (left). Quantification of DIAP1 levels achieved upon posterior-downregulation of nemo, measured as normalized posterior-to-anterior levels and compared to similar expression of GFP. Data are present as mean ± sem, n=8 discs. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.D. Yorkie is epistatic to Nemo: co-overexpression of the constitutively active mutant yki[S168A] together with nmo-RNAi produces typical yki[S168A] phenotypes: severe overgrowth of the affected area with concomitant increase in yki target gene expression (DIAP1). Due to lethality of the hh-Gal4-driven co-overexpression, another posterior driver en-Gal4 was used instead; anti-Hh staining (green) was used to mark the posterior compartment (left panel). Quantification posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 and UAS-yki[S168A]; en-Gal4, presented as in (C); n= 3-6 discs. Posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 are also significantly different from control UAS-GFP; en-Gal4 discs (right panel). Data are presented as mean ± sem. ***P < 0.005. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel).E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel). Lysates were immunoblotted with antibodies indicated in the figure (right-bottom panel). Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 200 μm.