Figure 3.

I870R is selectively nonresponsive to RhoA in vitro but otherwise is similar to wild-type PLD1. Glu–Glu tagged wild-type, I870R and PIM87/I870R PLD1 proteins were expressed in sf9 cells using the baculoviral system and were immunopurified. PLD activation was determined for 30 ng of each PLD protein using the head-group release assay in the presence of maximally effective concentrations of ARF1 and RhoA. Note that the maximal response of wild-type PLD1 to ARF1 is severalfold stronger than to Rho, as reported previously (Hammond et al., 1997). The experiment was conducted in triplicate. The values shown reflect activity after the subtraction of basal activity and assay background, and represent one of three experiments performed.