Fig. 1.

Kv1.2 phosphorylation in mammalian brain and heterologous cells. (A) RBM and lysates from cells expressing Kv1.2 alone or Kv1.2 plus Kvβ2 were digested without (−) or with (+) AP, subjected to SDS/PAGE, and immunoblotted using a general Kv1.2-specific mAb (K14/16). Numbers on the left refer to molecular weight of prestained molecular weight standards. (B) Identification of phosphorylation sites on Kv1.2 in rat brain using LC-MS/MS. A doubly charged, doubly phosphorylated peptide at m/z 573.035, derived from Kv1.2 purified from rat brain, was fragmented to produce this MS/MS with y- and b-ion series that described the sequence IPpSpSPDLKK (amino acids 438–446). The phosphorylation sites were unambiguously assigned to S440 and S441 because of mass assignments from β-eliminated y6, y7, b3, and b4 fragment ions with neutral loss of phosphoric acid H₃PO₄ and 2H₃PO₄.