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After being washed three times (10 min each) with PBST (PBS containing 0.05% Tween-20), the oocytes were incubated with a fluorescently labeled secondary antibody at room temperature for 45 min

The charged residues of peptide (such as Arg/Lys) form a salt bridge with the acidic residues on the enzyme surface (Glu256, Asp322), which induces the conformational change of the active center and destroys the stable coordination of copper ion ( In addition, peptides (such as FRAF) form a multi-level inhibition network by targeting MITF, TYR and TYRP1/2, and its mechanism goes beyond single target intervention: FRAF forms hydrogen bonds with ASP431 and ALA384 of MITF, blocking the combination of MITF and target gene promoters (such as TYR and TYRP1) and inhibiting the expression of melanin synthesis-related enzymes

We verified this calculation in two ways