Western Blot Analysis of CCL5-Stimulated hBMECs

Western blotting was performed as previously described (93 (link)). hBMECs were starved overnight and collected or stimulated with 100 ng/ml CCL5 for 10 min before harvest. Cells were washed with phosphate-buffered saline (PBS) and lysed in 1% NP-40 buffer with a protease inhibitor cocktail (Sigma) as previously described (12 (link)). Total protein levels were determined in a bicinchoninic acid assay (Thermo Fisher), and proteins were resolved by SDS–12% PAGE, transferred to nitrocellulose, blocked in PBS–1% bovine serum albumin (BSA), and incubated with antibodies in a blocker. Proteins were detected using horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham) and the Luminata Forte HRP substrate (Millipore).