Fig 1: FAT1 interacts with E3 ligases MIB2 to promote YAP/TAZ protein degradation.a, b HUVECs stably expressing flag-tagged control peptide (control) or flag-tagged intracellular domain of FAT1 fused to the transmembrane domain of the IL-2 receptor (FAT1ICD) were transfected with control siRNA or siRNA directed against endogenous FAT1, and cell proliferation (a) or YAP and TAZ protein levels (b) were determined (mean values ± SEM, n = 10, two-way ANOVA and Bonferroni’s post hoc test (a); 1 representative of 3 independently performed experiments (b)). c Proteins immunoprecipitated with an anti-FLAG antibody from lysates of HUVECs expressing Flag-tagged FAT1ICD or control were analyzed by mass spectrometry. Shown is a volcano plot highlighting enriched proteins in the FAT1ICD interactome. Statistical evaluation was two-sided Bayesian moderated t-test provided by the limma package. The p values were adjusted for multiple hypothesis testing using the method by Benjamini–Hochberg. d FAT1 was immunoprecipitated from HUVECs transfected with control siRNA or siRNA directed against FAT1, and immunoprecipitates were analyzed for presence of MIB2 (IgG: control immunoprecipitation; shown is a representative of three independently performed experiments). e, f HEK293 cells were cotransfected with HA-tagged MIB2 and the indicated Flag-tagged FAT1-ICD mutants (e) or with Flag-tagged FAT1 and the indicated HA-tagged mutants of MIB2 (f). Thereafter FAT1 (e) or MIB2 (f) was immunoprecipitated (IP) using anti-Flag or anti-HA antibodies, respectively, and precipitates were analyzed with the indicated antibodies (IB). Shown are a representative of three independently performed experiments. g, h The purified FAT1ICD-GST fusion protein was incubated with purified MBP-tagged MIB2 or His-tagged YAP protein followed by GST pull-down (g) or purified MBP-MIB2 fusion protein was incubated with purified FAT1ICD-GST and His-tagged YAP protein followed by MBP pull-down (h). Precipitated proteins were analyzed by immunoblotting (IB) using the indicated antibodies. Shown are a representative of three independently performed experiments. i HUVECs were transfected with control siRNA or siRNA directed against FAT1 or MIB2, and YAP/TAZ were immunoprecipitated (IP). Unrelated IgG served as an IP-control. Ubiquitination of YAP/TAZ was analyzed by immunoblotting (IB) using an anti-ubiquitin antibody. Immunoblots of the lysates (input) using the indicated antibodies are shown below (1 representative of 3 independently performed experiments).