Figure 5.

NPPD is a selective inhibitor of Kal/Trio GEF1 activity. (A) NPPD inhibits Kalirin and Trio GEF1 activity in cells. HEK293-derived pEAK Rapid cells were transfected with vectors encoding GFP, GFP-Kal-GEF1, or GFP-Trio-GEF1. For each transfection, duplicate wells were incubated with vehicle or 100 μM drug for 1 h, after which cells were lysed and processed for effector binding Rac activation assays (Schiller et al., 2005). Active (GTP-bound) and total Rac1 in lysates were detected by immunoblotting. (B) Lysates of pEAK rapid cells transfected with constructs encoding the indicated isolated GEF domains were incubated with GST-Rac1 loaded with GDP in the presence of 50 μM NPPD or an equal amount of vehicle (DMSO). GEFs bound to GST-Rac1 immobilized to glutathione-Sepharose beads were eluted and detected by immunoblotting with anti-Myc and anti-HA antibodies. In both A and B, signals (left panels) were densitized and plotted (right panels). One experiment performed in duplicate is shown (bars, range of variation; where not visible, the variation was small). The experiments were repeated with similar results.