Fig. 7. The GEF activity of Kalirin is increased following calyculin A treatment of cells. Activation of Rac was assessed using GST-Pak-Crib. (A) pEAK RAPID cells transfected with parent vector or vectors encoding KGEF1-7end or KEGF1-7end T/A were untreated (Con) or treated with 10 µM roscovitine (Ros) for 4 hours or 25nM calyculin A (CA) for 30 minutes. Transfected proteins were detected with Myc antibody (upper); phosphorylated protein was detected with Thr1590-P Ab (second row), and total Rac (third row) was visualized with Rac antibody. GTP-bound Rac was isolated using GST-Pak-Crib resin and quantified by western blot analysis with Rac antibody (bottom). (B,C) pEAK RAPID cells transfected with vectors encoding Kalirin-7 (B) or Kalirin-7 (C) were analyzed as described in A. Control cells were transfected with parent vector. Pooled cell extract incubated with GDP or GTPS, negative and positive controls. (D) Data from three independent assays. Activated Rac in control cells expressing Kalirin-7 (upper) or Kalirin-7 (lower) set to 100%; activated Rac in roscovitine or calyculin-A-treated cells expressed as a percentage of relevant control; P values were calculated using Student's t-test; ^*P<0.05.