PEDF in plasma is a phosphoprotein. (A) Aliquots of rPEDF, plPEDF active (phosphorylated) ERK, phosphorylated α-casein (phos cas), and dephosphorylated α-casein (dephos cas) were subjected to 10% SDS-PAGE (1 μg/lane) and immunoblotted with antiphospho-Ser, -Thr, or -Tyr Abs in the presence or absence of the appropriate phosphorylated amino acid (2.5 mM). As a control, the samples were blotted with anti-PEDF, anti–phosphorylated ERK (αpERK), and anti–general ERK (αgERK) Abs, or stained with gel code for the α-casein. (B) rPEDF (2 μg) or plPEDF (2 μg) were incubated with alkaline phosphatase (APase) conjugated to acrylic beads (2 U) or with protein A sepharose CL-4B (1.6 mg) for 45 minutes at 30°C. Following incubation, samples were centrifuged in order to remove the phosphatase, and the supernatants were subjected to in vitro CK2 or PKA phosphorylation as described in “Materials and methods.” Phosphorylated products were analyzed by 10% SDS-PAGE and blotting, followed by exposure to autoradiography (Auto, top panel) and immunoblot with anti-PEDF Ab (bottom panel). (C) Quantitative analysis of the experiment depicted in panel B is presented as a mean ± SD (n = 4). (D) PEDF purified from human plasma was subjected to alkaline phosphatase treatment as described in “Materials and methods”. Thereafter, aliquots (1.5 μg) were incubated with fresh human plasma (8 μL) and [γ³²P]-ATP (10 μM, 6 Ci/mmol [222 GBq/mmol]) in the presence or absence of PKA inhibitor (PKI, 1 μg/mL) or heparin (100 μg/mL). Control samples were subjected to in vitro CK2 or PKA phosphorylation as described in panel B. Phosphorylated products were analyzed as described in panel B. Vn indicates plasma vitronectin.