Transcriptional regulation of cytokine-induced CD40 expression.

(A) Time-dependent effect of TNF-α (100 U/mL) plus IFN-γ (1000 U/mL) on CD40 mRNA expression in the cultured HUVECs (calculated as percentage of basal CD40 expression, n = 3-7; *P < .05 versus basal). The inset shows the effects of 100 U/mL TNF-α and 1000 U/mL IFN-γ on CD40 protein expression after 14 hours. Typical Western blot analysis is shown with the relative intensities, as judged by densitometry, indicated at the top. Qualitatively identical results were obtained with at least 2 further batches of HUVECs. Loading and transfer of equal amounts of protein in each lane were verified by reprobing the membrane with an anti–β-actin antibody. (B) PCR-based run-on analysis of the de novo expression of CD40 and EF-1 mRNA in isolated nuclei of cultured HUVECs that had been exposed to TNF-α (100 U/mL) plus IFN-γ (1000 U/mL) for 6 hours. The isolated nuclei were either lysed immediately (0 min) or incubated for 30 minutes at 30°C. The figure depicts the result of one experiment; a qualitatively identical result was obtained in another experiment with a different batch of cells. (C) Effects of IFN-γ (1000 U/mL) or TNF-α (1000 U/mL) on nuclear translocation of IRF-1 (1 constitutive and 2 inducible complexes, as indicated by the arrows) in the cultured HUVECs, and effect of cycloheximide (Cx; 10 μM) on IFN-γ (100 U/mL)–stimulated nuclear translocation of IRF-1. Typical EMSA is shown with nuclear extracts obtained after 3 hours of exposure of the HUVECs to the cytokines. Qualitatively identical results were obtained with at least 4 further batches of HUVECs.