Stx1B and Stx2B activate RhoA and increase endothelial monolayer permeability. (A) Confluent HUVECs were treated with Stx1B or Stx2B for 5 or 15 minutes. Cells were then fixed, permeabilized, and stained for actin (green) and vinculin (red) as a marker for focal adhesions. Bars, 10 μm. (B) Confluent HUVECs on transwell filters were treated with 200 ng/mL Stx1B or Stx2B for 15 minutes or 1 hour. Monolayer permeability was measured by the penetration of FITC-dextrin to the bottom part of the transwell chambers. Results are shown as the mean ± SE for 5 independent experiments. Stx2B increased trans-endothelial permeability more than did Stx1B at both time points (P < .05). (C) HUVECs were treated with Stx1B or Stx2B for 10 or 30 minutes before lysis buffer was added. Cell lysates were probed for total RhoA or GTP-bound RhoA. The experiment was performed 3 times with similar results.