Figure 8. Rap2 colocalize with Ccdc124 and RasGEF1B at the subcellular level.

Subcellular localizations of endogenous Rap2 and Ccdc124 or RasGEF1B proteins were studied in HeLa cells by immunofluorescence methods. (A) At anaphase, Rap2 was clearly localized at the midzone, while Ccdc124 concentration at the same localization was less pronounced. However, at telophase, both proteins were concentrated at the puncta characterizing the midbody, Rap2 rather surrounding Ccdc124. During cytokinetic abscission, a clear colocalization of both Rap2 and Ccdc124 were observed at the midbody. (B) Similar to panel (A), Rap2 translocation to the midzone has started during anaphase. Two representative images of anaphase cells were shown in the corresponding panel. Both Rap2 and RasGEF1B proteins colocalized at the midbody during cytokinetic abscission. Arrowheads either indicate subcellular localization of Rap2 at anaphase and telophase, or they indicate the colocalization of Rap2 with RasGEF1B/Ccdc124 at the midbody during cytokinesis. (C) HeLa cells were transfected with shRNA vectors (Sh1, Sh2, Sh3) targeting Rap2, then cell lysates were collected at 48 hrs post-transfection, and immunoblotted with anti-Rap2 Ab. Scrambled control transfection was indicated (Scr). Calnexin expression was monitored as loading control. In parallel experiments, similarly treated cells were immunostained with anti-Rap2 and anti-α-tubulin antibodies, followed by scorings for multinucleation (n = 5± SD), as reported on the graph above the immunoblot. (D) Representative micrographs of midbody stage cells depleted in endogenous Rap2 (C). Bars represent 10 µm.