Fig 4.

(A) Internalization of surface-biotinylated JAM-A protein. Confluent mBMECs treated with and without cycloheximide (CXD) were surface biotinylated at 0°C and then exposed to CCL2 for 60 min at 37°C to allow internalization. Any membrane-bound biotin was removed by glutathione solution (glutathione stripping [gs]). Lanes 1 and 2, biotinylated JAM-A at the cell surface; lane 3, glutathione stripping of surface biotin; lanes 4 and 5, total cell lysate; lane 6 and 7, portion of biotinylated-internalized proteins. Adjusted blot showing the time course (0 to 60 min) of internalized biotinylated JAM-A during mBMEC exposure to CCL2. (B) Quantification of internalized JAM-A during the exposure to CCL2 and opening of the brain endothelial cell barrier. The percent internalized protein was estimated as the percentage of the total biotinylated JAM-A. GADPH (glyceraldehyde-3-phosphate dehydrogenase) represents an internal loading control. Data represent averages ± SDs of 5 independent experiments.