Scambio interacts specifically with the constitutively activated forms of Rac1, Rac2, Rac3 and Cdc42, but not with V14RhoA (A)CHO-K1 cells were transfected with HA-Sabi and GST only or GST-tagged wild type Rac3, N17Rac3, or V12Rac3. Scambio was immunoprecipitated using monoclonal anti-HA antibodies and complexed GST-tagged Rac3 mutants were detected using polyclonal Rac3 antibodies. Western blot analysis is shown of 7.5 μg of lysates (right panel) or immunoprecipitates from 0.4 mg of lysates (left panel). (B) COS-1 cells were transfected with HA-Scambio and the indicated constitutively active, GST-tagged small GTPases. GST-tagged proteins were pulled down using glutathione-agarose beads. Scambio was detected using anti-HA antibodies and GST-tagged GTPases using anti-GST antibodies. Western blot analysis is shown of 10 μg of lysate (right panel) or pull-down from 0.5 mg of lysate (GST-PD, left panel). The sizes of RhoA and Cdc42 are larger because these proteins also contain a Myc tag.