Fig. 7.

The Hippo signaling pathway is affected in patient fibroblasts. a Quantification of the endogenous TAZ (***P < 0.001), YAP (**P < 0.05) and MST1 transcript levels in control and patient fibroblasts by quantitative RT-PCR. b YAP and TAZ in whole cell lysates obtained from control and patient cell lysates. Probing was with YAP/TAZ-specific antibodies (YAP ~70 kDa; TAZ ~50 kDa) and GAPDH-specific antibodies (GAPDH ~38 kDa) as loading control. c MST1 protein levels appear unaltered in patient cells. Antibodies specific for CDK5RAP2, MST1 and GAPDH were used. d YAP and TAZ distribution in control and patient fibroblasts. Immunofluorescence analysis was carried out with YAP/TAZ antibodies. Scale bar 10 µm. e Hippo pathway genes are downregulated in patient cells as shown by qRT-PCR analysis (left panel) (P values, *<0.05, ***<0.001). The increase in BIRC5 transcript levels leads to increased protein amounts (right panel). f Co-immunoprecipitation assays to study the interaction of CDK5RAP2 with TAZ. Myc-tagged CDK5RAP2 full length, and the C1 and the C2 proteins were tested. HEK293T cells were transiently co-transfected with Flag-tagged TAZ and Myc-tagged CDK5RAP2 proteins. Immunoprecipitation (IP) was done with Flag-specific beads, the resulting blots were probed with mAb 9E10 to reveal Myc-tagged and pAb anti-Flag to reveal FLAG-tagged proteins. FLAG-hnRNPF was used as control. g Effect of ectopically expressed Myc-CDK5RAP2 on endogenous TAZ mRNA levels as revealed by qRT-PCR to analyze the transcript abundance of endogenous TAZ in untransfected HEK293T cells (WT) and in cells ectopically expressing Myc-CDK5RAP2 (***P < 0.001). The mRNA reduction leads to decreased protein level (panel above the graph). HEK293T cells were transfected with plasmids coding for Myc-CDK5RAP2 and Flag-TAZ as indicated