Figure 2. Abba interacts with and activates Rac1.

(A) Abba interacts with Rac1. 3T3 cells were co-transfected with Myc-Rac1 and Flag-Abba (lane 1), Myc-Rac1 alone (lane 2) and Flag-Abba alone (lane 3). After transfection, the cell lysates were immunoprecipitated with Myc antibody, and Rac1-associated Flag-Abba proteins in the pellets were measured by Western blot using Flag antibody. The same blot was re-probed with Myc antibody to show equal immunoprecipitation. The expression level of Flag-Abba in the transfected cells was determined by immunoblotting of whole cell lysates (WCL) with Flag antibody as shown in the bottom panel. (B–C) Analysis of the effect of overexpression of GFP-Abba on Rac activation in response to PDGF. Cells were starved at 0.2% serum-containing medium for 24 h and stimulated with PDGF for 10 to 60 min. GTP-bound Rac (GTP-Rac) in cell lysates was determined by pull-down with GST-PAK-CRIB followed by Western blot with Rac1 antibody. Aliquots of the cell lysates were also immunoblotted with Rac antibody as the input control. The chart represents the normalized levels of GTP-Rac to the input Rac1, and error bars were standard derivations of three independent experiments. (C) Cells co-expressing GFP-Abba with Myc-Rac1 or with Myc-Rac1^G12Vcells were incubated in 0.2% serum-containing medium for 24 h and stimulated with PDGF for 30 min. The lysates derived from treated cells were immunoprecipitated with Myc antibody, and the pellets were immunoblotted with GFP and Myc antibody, respectively. Aliquots of cell lysates were also blotted with α-tubulin antibody for the loading control. (D) Cells stably expressing Myc-Rac1^G12V (a and b) or Myc-Rac1 (c and d) were transiently transfected with GFP-Abba, and stained with phalloidin (a and c) and GFP antibody (b and d). A cell co-expressing GFP-Abba and Myc-Rac1^G12V developed prominent ruffles. Scale bar: 10 μm.