GTP-bound GTPases were isolated as in (107). Briefly, GST-PAK-PBD (a gift from K. Burridge) was expressed in Escherichia coli strain BL21(DE3), conjugated to Glutathione Sepharose 4B beads (GE Healthcare), and stored for up to 2 weeks at 4°C. Cell lysates were mixed with the GST-PAK-PBD beads for 45 min rotating at 4°C, followed by two washes with 1× ice-cold lysis buffer, and resuspended in sample buffer for SDS-PAGE.

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