Fig. 3: GEF activity of Vav1 mutant.
From: Vav1 mutations identified in human cancers give rise to different oncogenic phenotypes
The capacity of the Vav1 mutants E59K, D517E, and L801P to function as GEFs for Rac1 was analyzed. NIH3T3 cells stably expressing pcDNA3, Vav1, oncVav1, E59K, D517E, and L801P were transfected with FLAG epitope-tagged Rac1. Starved cell lysates were then incubated with the bacterial fusion protein that expresses Pak immobilized on glutathione-Sepharose beads. Bound proteins were separated on SDS−PAGE and immunoblotted (WB) with anti-FLAG mAbs, as indicated. Expression of transfected Vav1, Rac-FLAG, and actin was detected as indicated. The figure depicts one representative experiment of two experiments performed. Numbers outlined between the rows refer to the extent of Rac activation, calculated by the intensity of Rac-GTP bound to GST-Pak divided by the level of total Rac-Flag in each sample. The level of Rac activation by Vav1 was specified as 1
