Active GTPase Assays

GTPase activation assay was performed using Cdc42 G-LISA kits following manufacturer’s instructions (Cytoskeleton) as well as described elsewhere (22). Briefly, Cell lysates (1,5 × 10⁵ cells x triplicate) were prepared using G-LISA lysis buffer, snap-frozen in liquid nitrogen and processed within 2 weeks after preparation. Lysate aliquots, corresponding to Cdc42 assay, were applied, respectively, to wells coated with Cdc42-GTP-binding protein. Active, GTP-bound GTPases bound to the wells were detected with the corresponding specific Ab. Constitutively active proteins were used as a standard.