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Fig. 6. MIM-B binds to Rac. (A) MIM-B binds Rac through its IMD. Binding to GTPase mutants was determined by GST pull down. Lysates of COS-7 overexpressing myc-MIM-B, myc-MIM-B {Delta}IMD, myc-MIM-B K4D, myc-IMD or myc-IMD K4D were incubated with recombinant GST-N17 or -L61-Rac or -Cdc42 in low-salt conditions and binding was determined by immunoblotting with anti-myc antibody. L, lysate. (B) Endogenous MIM-B binds to Rac. Representative pull down experiments performed as described above in normal salt conditions using mouse brain extract as protein source. (C) The MIM-B IMD directly binds to Rac mutants. (Left) Recombinant His-tagged IMDs used for pull-downs with recombinant GTPases were separated on SDS-PAGE and stained with Coomassie Blue. (Right) Recombinant GST-GTPases were incubated with either purified His-IMD wild-type (wt) or K4D and binding was revealed by SDS-PAGE and Coomassie Blue staining.