Endothelial NF-κB is activated by angiogenic inhibitors. (A) Protein-DNA array data expressed as the relative signal intensity of transcription factor (TF) binding in human microvascular endothelial cells (HMVECs) activated with vascular endothelial growth factor (VEGF; 1 ng/mL, gray bars) and VEGF plus pigment epithelial-derived factor (PEDF; 20 nm, black bars): the arrows indicate transcription factors altered more than 2-fold. (B) Nuclear localization of NF-κB in HMVECs treated with VEGF (200 pg/mL), TSP1 (100nM), and PEDF (20nM) as indicated, fixed, and stained for p65. Images were viewed with a Nikon Eclipse TE 2000-U microscope with a 40×/0.60 air objective and Fluoromount G imaging medium (Southern Biotech), and captured with a Nikon LH-M100 C-1 camera. Metamorph, Adobe Photoshop CS3 Extended Version 10.01, and Corel Photo-Paint 10 software were used. (C) Quantitative analysis of the experiment in panel B. P value (determined by a 1-way analysis of variance) is shown where the difference with VEGF reached statistical significance. (D-E) Western blot analysis of NF-κB activation: nuclear extracts from HMVECs treated 30 minutes with indicated combinations of bFGF (10 ng/mL), VEGF (200 pg/mL), TSP1 (100nM), and PEDF (10nM) were probed for NF-κB p65; cytoplasmic extracts were probed for IκBα (D) or phospho-IκBα (E). To assess loading, the membranes were reprobed for TATA-binding protein (TBP) and tubulin. Tumor necrosis factor-α was used as a positive control. Three independent experiments were performed with similar results. (F) Time course of NF-κB activation was determined by measurements of IκB phosphorylation in ECs pretreated with proteasome inhibitor MG132 (30 minutes, 10μM). To quantify the results of the Western blot, we measured the phospho-IκB to GAPDH ratio and plotted it as a function of time on a linear scale. A representative of 3 independent experiments is shown.