Monoclonal antibody PHF‐1 recognizes tau protein phosphorylated at serine residues 396 and 404

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Abstract
The microtubule-associated protein τ is hyperphosphorylated in the paired helical filaments (PHFs) of Alzheimer's disease. Immunological and direct chemical studies have identified Ser396 and Ser404 as two of the phosphorylated sites. Previously, we have demonstrated, using synthetic τ peptides containing phosphorylated Ser396, that this site is recognized by the monoclonal antibody PHF-1. The present sudy extends this observation by showing that PHF-1 recognizes τ peptides containing either individually phosphorylated Ser396 or Ser404, but that there is a >10-fold increase in the sensitivity of detection of τ peptides by PHF-1 when both serines are phosphorylated. The recognition of singly or doubly phosphorylated Ser396 and Ser404 in τ by PHF-1 can also be demonstrated in Chinese hamster ovary cells transfected with full-length wild-type τ constructs or mutant constructs with Ala substituted for Ser396 or Ser404. We conclude that the PHF-1 epitope contains both phosphorylated Ser396 and Ser404.