We transfected HEK293 cells with GAB2 clones expressing either the S592F, P621fs, or R154Q mutations to assess their effects on RAS/MAPK and AKT signaling pathways. Site-directed mutagenesis (SDM) was used to generate the two missense mutations S592F and R154Q using the Agilent QuikChange Lightning SDM Kit, with primer pairs designed to introduce each mutation (primer sequences available on request). In brief, the GAB2 OmicsLink expression clone plasmid with CMV promoter (EX-N0090-M02, GeneCopoeia, Inc., Rockville, MD) was grown overnight at 37°C in LB broth containing ampicillin, and plasmid DNA extracted using the Qiagen Plasmid Midi prep kit. Mutagenesis of plasmid DNA was carried out for each mutation, followed by transformation of XL10-Gold ultracompetent cells, which were subsequently grown on LB-ampicillin agar plates and incubated overnight at 37°C. Single clones were picked from each plate and grown overnight at 37°C in LB broth containing ampicillin. Plasmid DNA was then extracted as above, and Sanger sequencing performed to confirm successful insertion of mutations.
A custom-designed plasmid containing the GAB2 coding sequencing with the frameshift P621fs mutation was synthesized using the GeneArt™ Plasmid Construction Service (Thermo Fisher Scientific).
A custom-designed plasmid containing the GAB2 coding sequencing with the frameshift P621fs mutation was synthesized using the GeneArt™ Plasmid Construction Service (Thermo Fisher Scientific).