Fig. 4

TAZ interacts with c-Jun for Irs1 transcription. a Mouse gastrocnemius muscle genomic DNA was sheared by sonication, and chromatin fragments were immunoprecipitated with c-Jun antibodies. ChIPed DNA was analysed by qRT-PCR with primer sets flanking TBE1 (n = 3). Eight- to ten-week-old mice were used. b TAZ interacted with c-Jun. 293T cells were co-transfected with F-TAZ- and c-Jun-expressing plasmids. After immunoprecipitating with anti-FLAG antibodies, the eluted samples were analysed by immunoblotting with TAZ and c-Jun antibodies. c Endogenous c-Jun interacted with TAZ. C2C12 cell lysates were immunoprecipitated with IgG or c-Jun antibodies, and immune complexes were detected using TAZ and c-Jun antibodies. d Mutating c-Jun-binding sites in TBE1-luc decreased TAZ- and c-Jun-mediated reporter activity. The mutant c-Jun-binding site was generated by site-directed mutagenesis. Luciferase reporter plasmids harbouring wild-type (TBE-luc) or mutant c-Jun-binding sites (TBE1m-luc) were transfected into 293T cells together with c-Jun- and TAZ-expressing plasmids. Luciferase reporter gene activity was analysed 24 h after transfection (n = 3). e Wild-type (amino acids 1–395), N-terminus-deleted TAZ (amino acids 124–395 and 164–395) and WW domain-truncated TAZ expression plasmids (ΔWW) were transfected into 293T cells together with a c-Jun expression plasmid. Cell lysates were immunoprecipitated 24 h after transfection with FLAG antibodies, and the eluted samples were analysed by immunoblotting using TAZ and c-Jun antibodies. f The transcriptional activity of the TAZ deletion mutant was assessed using a TBE1-luc reporter plasmid. 293T cells were co-transfected with the TAZ deletion mutant together with a c-Jun expression plasmid as described in e. A Renilla luciferase plasmid was used for transfection normalisation. Cells were lysed, and luciferase activity was measured 24 h after transfection (n = 3). Data are presented as mean ± SD. Statistical analysis was performed using Student’s t-test. ns not significant; ***p < 0.005