PEDF increases the binding of NF-κB, p300, and histone acetylation while restricting NFAT binding at cFLIP promoter. (A) ChIP analysis of NF-κB binding to the cFLIP promoter: DNA-protein complexes from HMVECS treated as indicated were precipitated with p50 antibody and amplified with primers for the area flanking the κB-binding site. Immunoprecipitation with antibody to RNA polymerase II and IgG served as positive and negative controls, respectively. (B) HMVECs were treated with VEGF plus or minus PEDF, nuclear extracts precipitated with the antibodies for p65 (left) or p50 (right), and analyzed by Western blot for HDAC1, p65 and p50, respectively. The blots were assessed by densitometry and the ratio between HDAC1 and p65 or p50 calculated (see numbers above the panels). A representative of 3 independent experiments is shown. (C) ChIP was performed with p300 antibodies, followed by quantitative PCR with the primers for the same cFLIP promoter fragment as in panel A. (D-E) ChIP was performed with the antibodies for AcH3 (D) and AcH4 (E), followed by quantitative PCR with the same primers. (F) ChIP was performed with the antibodies for NFATc2 and quantitative PCR with the same primer set. Statistical significance was calculated by 1-tailed Student t test. P values reflect the comparisons of VEGF with VEGF plus PEDF and VEGF plus PEDF with VEGF plus PEDF plus BMS, respectively. (G) ECs were infected with AdC or Ad-SR, treated for 4 hours as indicated (1 ng/mL VEGF, 20 ng/mL PEDF), and compared with the untreated control. The results of the 2 independent experiments are shown (Ad-SR1 and Ad-SR2, respectively).