RUFY1 and RUFY3 interact with Arl8b, and RUFY1 localize to compartments positive for early/recycling endocytic markers. (A) HeLa cell lysates were immunoprecipitated with anti-Rab8 antibodies bound to Protein A/G beads. The precipitates were immunoblotted (IB) with the indicated antibodies. (B) Recombinant GST and GST-Arl8b proteins immobilized on glutathione-coated-agarose beads were loaded with either GTP or GDP and then incubated with HEK293T cell lysates expressing RUFY1-FLAG or RUFY3-FLAG. The precipitates were IB with an anti-FLAG antibody and Ponceau S staining was done to visualize the purified proteins. (C) Densitometric analysis of RUFY1 and RUFY3 pulldown (normalized to input signals) using GTP-loaded GST-Arl8b. The values plotted are the mean ± SD from three independent experiments (*P < 0.05; unpaired two-tailed t test). (D–J) Representative confocal micrographs of HeLa cells immunostained for endogenous RUFY1 and various endocytic markers (D) Rab14, (E) EEA1, (F) SNX1, (G) LAMP1, (H) Lysotracker Red (LTR), (I) CI-M6PR and (J) Vps26. In the inset, the arrowhead marks the colocalized pixels. Bars: (main) 10 µm; (insets) 2 µm. (K and L) Pearson’s and Mander’s colocalization coefficient quantification of endogenous RUFY1 with various indicated 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. Source data are available for this figure: SourceData FS1.