Functional cooperation between 3BP2, Vav proteins, and Rho GTPases during NFAT activation. (A) 3BP2 activates Rac1 in Jurkat T cells. Cells were transfected with the indicated plasmids. After 24 hours, active forms of HA-Rac1 were captured by incubation of lysates with a GST-PDB fusion protein and glutathione beads. After extensive washings, bound HA-Rac1 proteins were detected by anti-HA immunoblotting. Expression of total HA-Rac1 was assessed on aliquots of cell lysates. Fold stimulation of GTP-bound HA-Rac1 was determined following normalization against total HA-Rac1 (left panel). The average of 2 representative experiments is shown (right panel). Daudi cells were cotransfected with 5 μg NFAT luciferase reporter and HA-tagged 3BP2 in the presence of either (B) 30 μg Vav1 L213A (Vav1 DN) or Vav2 L212A (Vav2 DN) or (C) 30 μg Rho N19, Rac N17, or Cdc42 N17, and luciferase activity was measured. Values represent the mean plus or minus the standard deviation from triplicate samples. Samples from the same lysates were immunoblotted with anti-myc or anti-3BP2 antibodies (insets).