Transwell endothelial permeability measurements.

The Transwell permeability assay was done following the kit’s instructions (Cell Biologics, CB6929). Briefly, mLMVECs were transfected with CREB expression plasmid using lipofectamine 3000 regents for 2 days. Transfected cells were seeded on the 0.4 μm polycarbonate membrane of 6.5-mm Transwell inserts in a 24-well plate, and incubated for 3 days until full confluent monolayers were formed. IL-1β (2 ng/mL) and HRP (100 μg/mL) were added into starved cells in Transwell inserts filled with serum-free medium. Cells were incubated for the indicated time for permeability analysis. Media (20 μL) from the lower chamber was transferred to a 96-well ELISA plate. TMB substrate (50 μL) was added into the wells, and left for 5 minutes until the solution turned blue. Spectrophotometric absorbance analysis at 450 nm provided a quantitative evaluation of the amount of HRP that was leaked from the disrupted barrier of endothelial cells. Data are presented as mean ± SEM of at least 3 independent experiments performed in triplicate.