Arf6 is involved in StxB but not histamine-evoked WPB exocytosis. (A) HUVECs were treated with Stx1B or Stx2B for 5 minutes before lysis buffer was added. Cell lysates were directly probed for Arf6, or pull-down assay was conducted to concentrate GTP-bound Arf6. The experiment was performed 5 times with similar results. (B) HUVECs in 24-well dishes were transfected with wild-type or dominant-negative (T17N) Arf6, and cell-surface ELISA assays were conducted 72 hours later after StxB treatment. Values for each condition were normalized to the amount of VWF secreted by unstimulated cells. The experiment was repeated twice with similar results. (C-E) HUVECs were transfected with cDNA encoding EGFP, CFP-Arf6, or CFP-DN Arf6. Seventy-two hours later, confluent cells were stimulated for 1 hour with Stx1B (C), Stx2B (D), or histamine (E); washed; and stained with fluorescent VWF antibody. Numbers of WPBs per cell were counted for 10-30 cells from multiple optical fields. Experiments were performed 3 times with similar results. Pairwise comparisons with or without Stx1B or Stx2B are significantly different (P < .01), except for the dnArf6 and Arf6 conditions.