Proteomic analysis of Rab11 endosomes uncovers XK. (A) Schematic representation of two-step isolation of Rab11 endosomes. STHdhQ7/Q7 cells were transfected with pcDNA_3.1-6xHis-Rab11 to label endosomes. Homogenates were prepared using a ball-bearing homogenizer and centrifuged to remove nuclei and then incubated with Nickel resins. Eluates from Nickel resins were overlaid on a sucrose gradient and centrifuged to obtain membrane-bound His-Rab11 in pellets (endosomes). (B) Western blot analysis of eluates from nickel resins and ultracentrifuge pellets (membrane-bound His-Rab11) with indicated antibodies. Shown are blot analyses from one of the four individual endosomal preparations. The open arrowhead indicates a protein likely to be an isoform of XK, as signals for this protein were very low at the basal state and appeared to increase when different amounts of XK-expressing plasmids were transfected (Fig. S1). Star symbols indicate signals for exogenous and endogenous Rab11, which were still persistent during the incubation of the blots with anti-EGFP and secondary antibodies. (C) Membrane-bound His-Rab11 in ultracentrifugation pellets were resuspended in glutaraldehyde and dropped onto grids for electron microscopic analysis. Images in both panels showed profiles of tubulovesicular clusters, which are reminiscent of recycling endosomes in cells. Shown are electron microscopic images from one of four individual endosomal preparations.(D) Examples of known Rab11 interactors and/or known cargo proteins identified in the proteomic analysis of isolated Rab11 endosomes are shown. Shown are the proteins identified in all four endosomal preparations. Source data are available for this figure: SourceData F1.