CK2 and PKA in vitro phosphorylation of PEDF triple mutants. (A) Schematic representation of the localization of Ser24, Ser114, and Ser227. The indicated residues were mutated to Ala or Glu to form the various triple mutants. (B-C) rPEDF and rPEDF mutants (indicated) were phosphorylated by CK2 (B) or PKA (C) as described in “Materials and methods.” Reaction was arrested by sample buffer and phosphorylated products were analyzed by 10% SDS-PAGE that was followed by exposure to autoradiography (top panels) and immunoblotted with anti-PEDF antibody (bottom panels).