Figure 9
From: Pro-inflammatory endothelial cell dysfunction is associated with intersectin-1s down-regulation
Expression of myc-ITSN-1s in LPS-treated ECs restores endocytosis and inhibits both iNOS and mtNOS protein expression. A. Myc-ITSN-1s-transfected ECs, 48 h post-transfection, were exposed to 1 μg/ml LPS cells for 48 h and then subjected to biotin internalization. NeutrAvidin Alexa Fluor 594 staining revealed the rescued ability of ECs to internalized biotin (a2). An enlarged region of interest (boxed area in a2) is shown in a3. Control ECs subjected to biotin internalization and NeutrAvidin Alexa Fluor 594 are shown for comparison in a1. Bars: a1, a2, a3 - 20 μm. B. Degree of inhibition of caveolae-mediated uptake in myc-ITSN-1s transfected and LPS-treated ECs, by comparison to controls was evaluated by ELISA in 3 different experiments. Bars, ±SD. C. Total ECs lysates (70 μg total protein/lane) prepared from control, LPS-treated and ITSN-1s transfected and LPS-treated cells were analyzed by SDS-PAGE, electrotransfer to nitrocellulose membranes and Western blotting for NOS2 protein expression. Actin was used as loading control. D. Enriched-mitochondrial fractions of control, LPS-treated and myc-ITSN-1s transfected/LPS-treated cells were analyzed as above for mtNOS expression.