Fig. 1.

PA influences PLD2-mediated Rac2 activation in vivo and in vitro. Rac2 activation was measured by a p21 activated kinase binding domain (PBD) pulldown assay. PBD has affinity for Rac2-GTP but not for Rac2-GDP. (A,C) PBD pulldown assay in whole cells transfected with myc-PLD2-WT (A) or with purified, baculoviral myc-tagged PLD2 and HA-tagged Rac2 (C) in the presence of 1 or 300 nM PA (DOPA, a membrane-soluble form of PA, was used for the whole cell experiments). Expression of increasing PLD2 and Rac2 equal loading controls are shown in western blots (W.B.) with anti-PLD2 or anti-HA antibodies, respectively. (B,D) Densitometry line graphs corresponding to A and C, respectively. Results shown are representative of three independent experiments performed in duplicate.