Association of TRAF6/TβRIII is regulated by functional TβRII and TβRI.

<p>(<b>A</b>) Myc-TRAF6, HA-TβRIII, and/or Flag-TβRII were expressed in SNU638 human gastric cancer cells and treated with TGF-β1 for 60 min. Immunoprecipitation and immunoblotting were carried out to elucidate the requirement of TβRII for TRAF6/TβRIII interaction. (<b>B</b>) TRAF6 and TβRIII interaction in TβRI−/− MEFs in the presence or absence of exogenous TβRI expression. (<b>C</b>) TRAF6/TβRIII interaction in the presence of wild-type, kinase inactive (KR: K232R), and constitutively active (TD: T204D) TβRI in HEK293 cells. (<b>D</b>) Interaction between TRAF6 and TβRIII in the presence of IN-1130 (0.1 µM), a specific inhibitor of TβRI kinase. (<b>E</b>) Olink assay for TRAF6 and TβRIII interaction in the presence of TGF-β and/or IN-1130. Transfected cells were counterstained with Alexa 488-labeled HA antibody to HA-TβRIII (green), and the nuclei were stained with Hoechst 33342 (blue). Bar = 2.5 µm. (<b>F</b>) The number of <i>in situ</i> PLA signals (red blobs) per cell was counted by semi-automated image analysis. (<b>G</b>) Co-immunoprecipitation analysis was conducted to elucidate the function of TβRI in TRAF6/TβRIII interaction. TβRI mutants KR, TD, and TD (E161A) mutated in TRAF6 consensus binding motif in TβRI (TD) were used. The results are representative of three independent experiments.