To explore the role of individual amino acids in the binding of GATAD1 peptide (position 95-109) to 14-3-3 proteins, we conducted an alanine scanning experiment. Each amino acid surrounding the phosphorylation site within the peptide was systematically mutated to alanine, except for the original alanine residues, which were mutated to glycine. These mutant peptides were immobilized on a cellulose membrane and employed to capture proteins from Hek293T lysate through pulldown assays.