The interplay between the CK2 and PKA phosphorylations of PEDF. (A) rPEDF and S24,114E mutant (EE) predialyzed in the different pH buffers (indicated) were phosphorylated by PKA for 45 minutes at 30°C. Reaction was arrested by sample buffer and samples were subjected to 10% SDS-PAGE. The gel was stained with Coomassie blue (bottom panel), dried, and subjected to autoradiography (top panel). (B) The experiment in panel A was reproduced with rPEDF and S24,114E mutant (EE) that were heat treated at 56°C for 15 minutes prior to phosphorylation. (C) rPEDF and S24,114A mutant (AA) predialyzed in the different pH buffers (indicated) were phosphorylated by PKA for 45 minutes at 30°C with or without heat treatment (56°C for 15 minutes prior to the phosphorylation). Reactions were 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). (D) plPEDF, rPEDF, S227A, and S227E mutants were phosphorylated by CK2 for 45 minutes at 30°C with or without heat treatment prior to the phosphorylation. Phosphorylated products were analyzed as described for panel A.