mHEV cells were stably transfected with ZO-1-EGFP. A) ZO-1 examined by western blot for non-treated cells (NT), lipofectamine-treated cells, vector-treated cells and ZO-1-EGFP transfected cells. ZO-1 is 220 kD and ZO-1-EGFP is about 247 kD. Therefore, this results in a wider band in the western blot for the cells expressing ZO-1-EGFP. B) mHEVa cells, transfected with vector or ZO-1-EGFP, were Immunolabeled with anti-VCAM-1 or isotype control antibodies (Ab), or examined for ZO-1-EGFP expression by flow cytometry. C) Representative micrographs of ZO-1-EGFP transfected mHEV cells and co-localization (yellow) in junctions labeled with rat anti-mouse VCAM-1 and a secondary donkey anti-rat IgG Texas Red. N=3 experiments. D) Spleen leukocyte transendothelial migration at 15 minutes in a parallel plate flow chamber at 2 dynes/cm2. *, p<0.05 compared to controls. Data are from 3 experiments and are presented as mean ±SEM.