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To further purify FOXO4 FHD and p53 TAD, gel filtration chromatography was conducted using Hi-Load 16/600 superdex 200pg and 75pg (Cytiva) on AKTA pure and AKTA prime systems (Cytiva)

Afterward, all the systems were subjected to a NVT (constant number of particles, volume and temperature) equilibration phase for 125,000 steps, using the V-rescale temperature-coupling method, with constant coupling of 1 ps at 303.15 K (Bussi et al
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doi: 10.1053/j.gastro.2009.06.049