Figure 3.

Effect of hemoglobins carrying Providence mutation on human pulmonary arterial endothelial cells. HPAECs were exposed to ferrous forms of either HbA, Providence (βK82D), HbS, HbS Providence (βE6V/βK82D), or crosslinked Providence (rHb0.1/βK82D), at equimolar concentration (100 µM) for 24 h. (A,C) Cell lysates were immunoblotted with primary antibodies against HO-1, ferritin light chain (L-ferritin), toll-like receptor 4 (TLR4), and vascular cell adhesion molecule 1 (VCAM). Equal loading was confirmed by re-probing the blots against β-actin. All immunoblot panels shown are representatives of three separate independent experiments. (B) Densitometric analysis was done for the HO-1 expression vs. corresponding β-actin levels and the values represent the average ratio of band intensities (HO-1:β-actin), n = 3. * p < 0.05 vs. untreated control; ^# p < 0.05 vs. corresponding HbS.