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METHOD DETAILS Sample preparation FOXO4 FHD (95-195) was subcloned into the pET His6 Tobacco Etch Virus (TEV) LIC cloning vector (2B-T, obtained from Scott Gradia, Addgene Plasmid #29666) and transformed into BL21 (DE3) cells

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Persistent cloudiness or visible aggregation after gentle swirling indicates poor solubility at that pH and points toward acetic acid water