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Do not freeze it again, as the freeze-thaw cycle can degrade the peptide

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In order to avoid further HIF-1 degradation during whole cell lysates preparation, cells were lysed directly with NuPAGE LDS Sample Buffer (1X) (Thermo Fisher Scientific, San Jose, CA, USA) in the presence of protease and phosphatase inhibitors cocktail (Cell Signaling Technology, Danvers, MA, USA), similarly to ref