Bimolecular Fluorescence Complementation for Receptor Interaction

Bimolecular fluorescence complementation vectors were generated by gateway cloning with donor vectors containing Axl (pDONR223-Axl, Addgene plasmid 23945) or EGFR (pDONR223-EGFR, Addgene plasmid 23935).^{29 (link)} These were cloned into pDEST-ORF-V1 (Addgene plasmid 73637) and pDEST-ORF-V2 (Addgene plasmid 73638), respectively. An expression vector encoding full length Venus fluorescent protein was also utilised as control. For confocal microscopy, HEK-293T cells expressing an H2B-mCherry nuclear marker were grown on glass coverslips within a six-well plate. These were transfected with 500 ng of each vector (or Venus control) using Polyplus Jetprime and incubated for 16 h. The coverslips were prepared for confocal microscopy by fixation with 1% paraformaldehyde for 5 min at room temperature. For high-content analysis, HEK-293T cells expressing an H2B-mCherry nuclear marker were grown on Greiner CELLSTAR 96-well plates in phenol red free media (Thermo Fisher Scientific). Each well was transfected with 20 ng of each vector (or Venus control) using Polyplus Jetprime and incubated for 16 h, in the presence of absence of gefitinib at the concentrations indicated. Single cell fluorescence intensity was measured using the ArrayScan XTI Live High Content Platform (Thermo Fisher Scientific).