PKCα activation and Stx1B-induced VWF secretion. (A) HUVECS were transfected with PKCα siRNA or negative control siRNA oligonucleotides. Cell lysates were prepared after 72 hours and analyzed by 4%-12% SDS-PAGE and Western blotting to detect total PKCα and β-actin. (B) Secretion of VWF strings was assessed in HUVECs treated similarly. Values are shown as mean ± SE; *P < .00005 and NS indicates P = .4, by Student t test. (C) HUVECs were treated for 5 minutes with 5nM Stx1B or Stx2B, and cell lysates were prepared for 3%-8% SDS-PAGE and Western blotting with primary antibodies specific for PKCα phosphorylated on Thr638 or Ser497 and appropriate fluorescently labeled secondary antibodies. Blots were stripped and reprobed with antibody recognizing total PKCα.