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Fig. 1. Rap1 activation induced by overexpression of Chat. (A) A diagram of Chat variants used in this study. We used an N-terminally myristoylated membrane-targeted form of Chat (Myr-Chat) to evaluate the activation status of Chat. C-terminal deletion mutants (Chat-{Delta}CT and Myr-{Delta}CT) lacked the Cas association activity. (B) 293T cells were transiently transfected with the indicated Chat-related expression plasmid together with small GTPase Rap1. After 30 hours, the activation level of Rap1 was compared by a pull-down assay. The amount of pulled-down GTP-bound Rap1 (GTP-Rap1), expression levels of transfected Rap1 (Total Rap1) and Chat-derivatives (Anti-Chat) is shown. Overexpression of Chat increased the GTP-bound Rap1 compared with the control pCAX vector transfectant. Myr-Chat activated Rap1 more effectively than wild-type Chat. Chat-{Delta}CT and Myr-{Delta}CT abolished the Rap1 activation competence.