Arl8b and RUFY1 colocalize on a subset of early endosomes positive for Rab14. (A) Immuno-electron micrographs of ultrathin cryosections of HeLa cells transfected with Arl8b-HA (immunolabeled with 10 nm gold) and RUFY1-GFP (immunolabeled with 15 nm gold). Colocalization of RUFY1 and Arl8b was observed on smaller vesicles juxtaposed to endosomes (E, left and middle panels) and late endosomes/MVBs (right panel). Bars: 200 nm. (B–E) Representative confocal micrographs of HeLa cells transfected with Arl8b-HA and RUFY1-GFP and stained for various endocytic markers (B) Rab14, (C) EEA1, (D) CI-M6PR, and (E) Rab7. In the insets, the arrowhead marks the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (F and G) Pearson’s and Mander’s colocalization coefficient quantification of RUFY1-GFP and Arl8b-HA-positive compartments with indicated endocytic markers. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set.