Fig. 3. Analysis of phenotype induced by overexpression of PLD2, and localization of endogenous PLD2. (A) MDCK cells were infected with the adenovirus coding for wild-type or an enzymatically inactive mutant of PLD2 and photographed using phase contrast optics. The vector also codes for the green fluorescent protein, and the infected cells are shown. The graph shows PLD activities in infected cultures. Bars, 20 µm. (B) Cells were infected overnight with adenovirus coding for the indicated construct, or treated with C₈-PA for 5 hours and stained with Alexa Fluor^® 546 phalloidin or immunostained for E-cadherin. As in A, virtually all cells in the fields were infected. (C) Localization of PLD2. Corneal epithelial cells infected with adenovirus coding for PLD2(wt) or uninfected cells were stained with an anti-PLD2 antibody. Unspecific staining of uninfected cells was determined by pre-incubating the antiserum with the immunizing peptide (`blocked'). Uninfected cells 8 hours after induction of wounding were also stained (right panel). Arrows indicate staining of lamellipodia. The lamellipodial staining was blocked by preincubation of the antibody with the peptide (data not shown). A confocal section close to the tissue culture plastic is depicted, so the nuclei are not visible.