FIGURE 3.

Plekhg4 is a functional GEF. A, GTPase activation by Plekhg4. COS7 cells were transiently co-transfected with Rac1, RhoA, or Cdc42 (HA-tagged in the pKH3 vector) and the indicated GEF in the pcDNA3.1 construct. After serum deprivation for 24 h, cell lysates were subjected to the PBD assay as described under “Results.” Top panel, activated (GTP-bound) GTPase precipitated with GST-PBD, visualized after anti-HA immunoblotting. Lower panels, expression levels of the indicated proteins in cell lysates. Bait GST-PBD was visualized by Ponceau Red staining of the blotted membranes, and -fold activation was determined after computer densitometry after normalization to GEF expression. Data shown are representative of three independent experiments in which activation varied by ±15%. B, Plekhg4-GTPase association. GST fusion proteins of Rac1, Cdc42, and RhoA were overexpressed in E. coli, purified on glutathione affinity chromatography, and their nucleotide content manipulated as described under “Results.” Immobilized GTPase were incubated with lysates from HA-Plekhg4- or Dbl-expressing COS7 cells and washed, and associated GEFs were visualized by immunoblotting. GST-GTPases baits were visualized by Ponceau Red staining of the blotted membranes. Shown is a representative of three independent experiments.