Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40

: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti‐pTH32–47 antibodies with phospho‐ and dephospho‐Ser40 forms of TH protein and peptide TH32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody‐capture ELISAs, anti‐pTH32–47 was more reactive with the phospho‐TH than with the dephospho‐TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho‐TH forms. In western blots, labeling of the ∼60‐kDa TH band by anti‐pTH32–47 was readily detectable in lanes containing protein kinase A‐phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti‐pTH32–47. Similarly, anti‐pTH32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8‐bromoadenosine 3′,5′‐cyclic monophosphate or forskolin enabled removal of TH activity by anti‐pTH32–47. Lastly, in immunohistochemical studies, anti‐pTH32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion‐fixed rat brain.