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Fig. 5. MIM-B activates Rac through its IMD. (A) MIM-B overexpression activates Rac. Activity of endogenous Rac was determined by pull-down from lysates of COS-7 cells transfected with an empty vector (Ø) or constructs encoding myc-MIM-B, myc-MIM or an HA-tagged constitutively active form (DH-PH) of mouse Vav-1 (Vav) using GST-PAK-CRIB. Rac activation was revealed by immunoblotting with anti-Rac antibody. Expression of tagged proteins was subsequently determined by immunoblotting with anti-myc and anti-HA antibodies. (B) The IMD of MIM-B is necessary and sufficient to activate Rac. Rac activity from COS-7 cells overexpressing myc-MIM-B {Delta}IMD ({Delta}IMD), myc-MIM-B, myc-IMD or myc-MIM-B K4D (K4D) was determined as described above. (C) IRSp53 does not activate Rac. Rac activity of COS-7 cells expressing myc-IRSp53 (IRS) or myc-MIM-B was determined as above. These results are representative of at least three different experiments.