Effect of PKCα knockdown and dominant-negative RhoA on VWF secretion induced by Stx1B and Stx2B. (A) HUVECs were transfected with siRNA against PKCα or GFP (Ctrl). Whole-cell lysates were prepared 72 hours later, and PKCα level was assessed by Western blot analysis. The membrane was stripped and reprobed for β-actin. (B) HUVECs were transfected with siRNA against PKCα or GFP, and cell surface VWF was assayed by ELISA 72 hours later. Values for each condition were normalized to the amount of VWF secreted by unstimulated cells. The graph represents the results of 3 independent experiments. **P < .01. (C-D) HUVECs were transfected with cDNA encoding EGFP or EGFP-DN RhoA. Seventy-two hours later, confluent cells were stimulated for 1 hour with either Stx1B (C) or Stx2B (D), washed, and stained with fluorescent VWF antibody. Numbers of WPBs per cell (stimulated or unstimulated) were counted for 10-30 cells from multiple optical fields. Experiments were conducted 4 times with similar results. Pairwise comparisons with or without Stx1B or Stx2B are significantly different (P < .01) except for the dnRho conditions.