Single-Molecular Interaction Analysis Demonstrated That this S310F HER2 Mutant Formed Heterodimers with the EGFR Single molecule interaction analysis was performed to determine whether the S310F HER mutant formed a heterodimer with the EGFR. the phosphorylation of HER2 while it bound to wild-type HER2, EGFR-mediated phosphorylation is usually expected to occur around the S310F mutant. To confirm whether the S310F mutant HER2 retained its affinity to the EGFR, single molecule conversation analyses using TIRF microscopy were performed, which showed that S310F mutant successfully created complexes with EGFR. In KRas G12C inhibitor 4 conclusion, HER2 S310F mutant can form an active heterodimer with the EGFR and it can be inhibited by cetuximab, but not by trastuzumab in combination with pertuzumab. and the amount of protein in the supernatants was determined by a BCA assay (Pierce Biotechnology, Waltham, MA, USA). The proteins in the supernatants were separated by SDS-PAGE using 4%C12% bis-Tris gels (Invitrogen) as described above and transferred onto a nitrocellulose membrane. The membrane was blocked by pre-incubation in 5% BSA/0.2% TBST at room temperature for 30 min and then incubated with anti-EGFR antibody (Cell Signaling Technology), anti-phospho-EGFR (Tyr 1068) antibody (Cell Signaling Technology), anti-HER2 antibody (Cell Signaling Technology), anti-phospho-HER2 (Tyr 1221/1222) antibody (Cell Signaling Technology), anti-PARP (Cell Signaling Technology), anti-cleaved PARP (Cell Signaling Technology), or anti–actin antibody (Cell Signaling Technology) for overnight at 4 C. After washing three times with 0.2% TBST, the membrane was incubated with either HRP-conjugated goat anti-rabbit IgG Fc antibody (#31463; Thermo Fisher Scientific) or HRP-conjugated goat anti-mouse IgG antibody (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The membrane was washed three times with TBST, and the bound antibody was visualized by addition of Super Signal Pico West chemiluminescent substrate (#34080; Thermo Fisher Scientific) following the manufacturers instructions. The Image Lab program (BioRad, CA, USA) was used to determine the intensity of bands. 2.8. Single-Molecular Interaction Analysis Using Total Internal Reflection Fluorescence (TIRF) Microscopy Single-molecular interaction analysis was performed as described in a previous study with appropriate modifications [49]. Genes encoding fusion proteins composed of the extracellular domain of the EGFR fused with mCherry protein (EGFR-mCherry) and that of wild-type HER2 fused with eGFP (HER2-eGFP) were ligated into a bicistronic expression vector. STMN1 Another expression vector was prepared with the S310F mutant (S310F HER2-eGFP) replacing wild-type HER2.Then, these vectors were transfected into HEK293T cells (FreeStyle 293-T cells). After culture at 37 KRas G12C inhibitor 4 Cwith 5% CO2 for 1 day, the cells were dissolved in lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, KRas G12C inhibitor 4 10% glycerol) with protease (P8340; Sigma-Aldrich) and phosphatase (P5726; Sigma-Aldrich) inhibitor cocktail. After centrifugation at 15,000 for 10 min at 4 C, the supernatant was collected. Recombinant mCherry (4999-100; Biovision, Milpitas, CA, USA) and eGFP (4993-100; Biovision) protein standards were used to determine the concentration of fluorescently tagged proteins in the cell lysates. The fluorescence levels for recombinant eGFP and mCherry proteins of known concentrations (5C25 nM, five different concentration points) were measured using a fluorometer (Enspire 2300; Perkin-Elmer, San Jose, CA, USA) and were used to construct a calibration curve. Detailed procedures of the flow chamber construction are described in previous studies [50,51]. The flow chamber was washed twice with 200 L of PBS. Afterwards, 50 L of NeutrAvidin solution (A2666; Invitrogen) was added to the flow chamber and was incubated for 5 min at room temperature. Subsequently, the flow chambers were washed twice followed by addition of diluted biotinylated polyclonal RFP antibody (34771; Abcam, Cambridge, UK) for 5 min at room temperature. This antibody was reported to be reactive to all RFP variants from including mCherry. After the flow chambers were washed again, cell lysates were added and incubated for 10 min at room temperature. Finally, the flow chambers were washed with 0.1% lysis buffer/PBS. EGFR/HER2 heterodimers were then detected with a TIRF microscope equipped with.