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. 2013 Jul 19;8(7):e69289. doi: 10.1371/journal.pone.0069289

Figure 10. Rap2 effector proteins translocate to the midbody depending on the signal transduction activity of Rap2.

Figure 10

(A) HeLa cells were transfected with the vector containing GFP-labeled Rap Binding Domain of RalGDS [GFP-RBD(RalGDS)] which interacts only with the GTP-bound active form of Rap2, and cells were monitored at interphase and at cytokinetic abscission following immunostainings involving anti-Rap2 monoclonal Abs and Alexa568-red labeled anti-mouse secondary Abs. Colocalization of GFP-RBD(RalGDS) with Rap2 indicated that at the midbody Rap2 is in its active (Rap2.GTP) form. (B) HeLa cells were cotransfected with GFP-RBD(RalGDS) either together with HA-Rap2-WT, or with HA-Rap2-S17N (inactive dominant negative form) and their localizations were monitored with anti-HA-epitope Abs. Positioning of GFP-RBD(RalGDS) were assessed by monitoring GFP-signal observed at the midbody. Localization of the GFP-RBD(RalGDS) depended on the presence of active Rap2 in cells. As in (A), at least 50 cells were monitored in each experiment, and representative pictures display Rap2 and GFP-RBD(RalGDS) localizations observed at least ∼90% of cells cotransfected with indicated vectors. Bars represent 10 µm.