Pdest orf v2

SORL1 ORF was ordered from Origene in the pLenti-C-Myc-DDK plasmid (Origene, PS100064). The SORL1 ORF was digested using EcoRI and XhoI and ligated into the pLenti-C-GFP plasmid (Origene, PS100065) between EcoRI and XhoI restriction sites. pDEST-SorLA-v1 (Addgene, #154892) was generated by first PCR amplifying the SorLA coding sequence from the pLenti-SorLA-C-GFP vector using the primers 5’-GGTACTCGAGGCCACCatggcgacacggagcagcaggaggga-3’ and 5’-GGTCGAATTCggctatcaccatggggacgtcatctgaaaatccag-3’. PCR fragments were then subcloned into the pDEST-ORF-v1 (a kind gift from Darren Saunders, Addgene, #73637) [32 (link)] vector using the XhoI/EcoRI restriction enzymes. For the pDEST-ERBB2-v2 (Addgene, #154895), pDEST-ERBB3-v1 (Addgene, #154893) and pDEST-ERBB3-v2 (Addgene, #154894) vectors, LR reactions (LR clonase II, ThermoFisher Scientific) were performed using the pDEST-ORF-v1 and pDEST-ORF-v2 (Addgene, #73638) [32 (link)] destination vectors, and pDONR223-ERBB2 (a kind gift from William Hahn & David Root, Addgene, #23888) [62 (link)] and pDONR223-ERBB3 (Addgene, #23874) [62 (link)] shuttle vectors. pENTR2b-mVenus was LR subcloned into pEF.DEST51 (ThermoFisher Scientific, #12285011) to generate the expression plasmid, pEF.DEST51-mVenus. All vectors were verified by analytical digests and sequencing.

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).