Fig. 1. Inflammatory cytokines inhibit endothelial cell proliferation and induce expression of GBP-1. (A) Proliferation assay with HUVEC and MVEC upon addition of AGF (VEGF and bFGF, 10 ng/ml each, except control) and increasing concentrations of IL-1β, TNF-α or INF-γ. (B) Northern blot (upper panel) and western blot (lower panels) analyses of GBP-1 expression in MVEC incubated with either bFGF (10 ng/ml), VEGF (10 ng/ml), IL-1β (20 U/ml), TNF-α (300 U/ml), IFN-γ (100 U/ml), or control medium for 5 h and 8 h, respectively. Polyclonal rabbit anti-GBP-1 peptide antibody (αGBP-1 pAb), polyclonal rabbit anti-GBP-1 antibody (αGBP-1 rAb), actin control (Actin). (C) Immunostaining for detection of GBP-1 expression in HUVEC stimulated for 8 h as described in (B). (D) Northern blot analyses of GBP-1 mRNA expression in MVEC after incubation for various periods of time with IL-1β; (E) in the presence or absence of cycloheximide (CHX, 50 µM) and IL-1β (20 U/ml, 5 h induction). (F) RT–PCR analysis of IFN-γ expression in HUVEC and HUT 78 lymphocytes stimulated for 5 h with IL-1β (20 U/ml) and phytohemagglutinin-L (PHA, 5 µg/ml), respectively. Actin mRNA was amplified as a control.