siRNA-mediated down-regulation of PLD isoforms does not affect the number or appearance of WPbs. HUVECs were transiently transfected with either nontargeting shRNA plasmids or shRNA plasmids targeted at PLD1 or PLD2. (A) Forty-eight hours after transfection, PNS were prepared and analyzed by Western blot analysis using rabbit polyclonal anti–PC-PLD1 and anti–PC-PLD2 as well as monoclonal anti-vimentin antibodies (loading controls). (B) Quantification of PLD1 and PLD2 down-regulation. Signal intensities were quantified by densitometric analysis using the ImageJ software (NIH). Statistical significance (*P < .01) of the results obtained from 3 independent experiments was evaluated by unpaired Student t test. (C,D) WPb distribution in shRNA-transfected cells. Transfected cells were identified by fluorescence of the plasmid encoded dsRed and WPbs were visualized by labeling with monoclonal anti-human VWF antibodies. Transfection rates typically were in the range of 70% to 90%. Images shown here were chosen to show nontransfected and transfected cells in the same frame. Note that the anti-VWF staining is not affected by PLD1 (C) or PLD2 (D) depletion. Bars represent 10 μm.