TGF-β-enhanced HER2 activity depends on TβRI kinase activity. (A) NMuMG cells were treated with 2 μM Lapatinib or 5 μM SB431542 (SB) for 2 h, and 100 pM TGF-β1 was added in the last 1 h before being harvested for anti-HER2 pulldown and then for anti-phospho-tyrosine immunoblotting. (B) HEK293T cells were transfected with indicated plasmids before being harvested for anti-Myc pulldown and then for anti-phospho-Tyrosine immunoblotting. (C) NMuMG cells were treated with 5 μM SB431542 for 2 h, and 100 pM TGF-β1 was added in the last 1 h. Cells were then harvested for immunoblotting. (D) R1B/L17 cells overexpressed with empty vector or TβRI were treated with 100 pM TGF-β1 for 1 h before being harvested for immunoblotting. (E) SPR analysis of TβRI WT ICD—HER2 ICD and GGD ICD—HER2 ICD interactions. HER2 ICD protein was immobilized on Series S sensor chips CM5, and the binding experiments were carried out using a Biacore S200. The estimated Kd values were derived by fitting the association and dissociation signals with a 1:1 (Langmuir) model using the Biacore S200 Evaluation Software. The band intensity of phosphorylated proteins was normalized to total proteins. Source data are available for this figure: SourceData F2.