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Mixtures were incubated with Glutathione Sepharose 4 Fast Flow resin (Cytiva) pre-equilibrated in Binding Buffer for 1 h at 4 C with overhead rotation in a 1 mL spin- column (MoBiTec)

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The lysates were centrifuged (10.000 g , 30 min, 4 C) and the supernatants were loaded on equilibrated Ni-NTA agarose columns (Qiagen)

The NHE7-TFEB/TFE3 mechanism likely contributes to the observed induction of autophagy triggered by hypertonic stress [56][57] and may synergize with the increased synthesis of phosphatidylinositol-3,5-bisphosphate, a lipid crucially required for lysosomal proteolysis, in yeast [58][59] and mammalian cells challenged with hyperosmotic conditions [60]