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Protein-lipid overlay assay After 24 h of transfection, N2a cells were washed twice with TBS (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol) and cell extracts were prepared as explained above in TBS supplemented with 0.5% Tx-100 and protease inhibitors

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Enriched mutations from earlier rounds were retained (Supplementary Table 2)