Bilirubin induces DAF expression on vascular ECs. (A) HUVECs were treated for up to 8 hours with bilirubin (10 μM). Total RNA was isolated; Northern blots prepared and probed for DAF mRNA. The fold change in DAF mRNA was calculated after densitometric scanning of each band, normalized with respect to the ethidium bromide–stained 28S bands. (B) HUVECs were treated with bilirubin (0-10 μM) for 24 hours, before lysis and analysis of DAF expression by immunoblotting. The fold change in DAF expression was calculated by densitometry, normalized with respect to α-tubulin (n = 3). (C) HUVECs were treated with bilirubin (up to 10 μM) or vehicle alone for 24 hours before analysis of DAF expression by flow cytometry using mAb 1H4. Results are percentage increase in RFI above control ECs treated with plain EC culture medium (mean ± SEM; n = 6 experiments). *P < .05; **P < .01. (D) HUVECs were exposed to 95% air plus 5% CO₂ or 94% air, 5% CO₂ plus 1% CO (corresponding to 10 000 ppm of CO) for up to 8 hours, before lysis and quantification of DAF expression by immunoblotting with mAb IA10. The membrane was stripped and reprobed with antitubulin. Relative levels of protein expression were quantified using image analysis and expressed as percentage above air-treated control.