SHP2D61G-HA was amplified by PCR using the following primers: 5′-cccgctagcgccaccatgacatcgcggagatgg-3′ and 5′-atggcgcgcctcaagcgtaatctggaacatcgtatgggtatctgaaacttttctgctgttg-3′. The sequence for the HA tag is underlined. The PCR product was digested with Nhe I and Asc I and ligated into the pAAV-EF1a-DIO-EYFP-WPRE plasmid. GAB1Y627F, GAB1WT, and GRB2-GFP were also digested with Nhe I and Asc I from the cytomegalovirus (CMV)–GAB1Y627F and pcDNA5/FRT/TO-GRB2-GFP plasmid and ligated into the pAAV-EF1a-DIO-EYFP-WPRE plasmid. CMV-GAB1Y627F was a gift from A. Yart (INSERM) (35). pcDNA5/FRT/TO-GRB2-GFP was a gift from Y. Ye (Addgene plasmid number 86873; http://n2t.net/addgene:86873; RRID: Addgene_86873) (54). AAV was prepared as previously described (55). For AAV packaging, HEK 293T cells (1.2 × 107) were plated on 150-mm culture dishes (Thermo Fisher Scientific 157150) with 15-ml D10 culture medium [Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, SH30243.01) + 10% fetal bovine serum (Thermo Fisher Scientific SH30919.03)] in an incubator at 37°C under 5% CO2 for 24 hours. Thirteen micrograms of p5E18-RxC1 plasmid and 26 μg of pAd-ΔF6 plasmid with 13 μg of pAAV plasmid (EYFP, SHP2D61G, GAB1Y627F, GAB1WT, and GRB2-GFP) were transfected into HEK 293 T cells by the CaPO4 transfection method. Cells were washed with DMEM 6 to 8 hours after transfection, and culture medium was replaced with 20-ml fresh D10 medium. After 72 hours, culture medium was harvested for AAV purification. Solutions were stacked in order (from top to bottom) in an ultracentrifuge tube (Beckman Coulter, 324214) as follows: culture medium from each dish, 6 ml of 15% iodixanol (OptiPrep; Axis-Shield, 1045) solution [1 M NaCl, 1 mM MgCl2, 2.5 mM KCl, and 25% OptiPrep in phosphate-buffered saline (PBS)], 5 ml of 25% iodixanol solution (1 mM MgCl2, 2.5 mM KCl, 0.2% phenol red, and 42% OptiPrep in PBS), 5 ml of 40% iodixanol solution (1 mM MgCl2, 2.5 mM KCl, and 67% OptiPrep in PBS), and 4 ml of 60% iodixanol solution (1 mM MgCl2, 2.5 mM KCl, and 0.2% phenol red in OptiPrep). Tubes were centrifuged at 69,000 rpm at 18°C for 1 hour using a Beckman UltimaTL-100 K ultracentrifuge and a 70Ti rotor. About 4 ml of 40% iodixanol solution was harvested from the centrifuged column using a 5-ml syringe (KOVAX ND.SY1030-005). The harvested solution was mixed with 11 ml of PBS and filtered with an Amicon ultra-15 filter tube (Millipore, UFC910024), and the filter was washed twice with 15 ml of PBS. The remaining solution was harvested, and viral particles in the solution were quantified using quantitative PCR (qPCR).

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