Figure 2. Effect of Rab11 depletion on endocytosis.

Control and TbRab11 silenced cells were used for ligand binding and uptake assays as described in Materials and Methods. A. Binding (TL:A488 only) and uptake (Tf:A488 and TL:A488) of receptor-mediated endocytic cargo were measured by flow cytometry. Mean fluorescence intensities (MFI) normalized to control un-silenced cells are presented (mean ± std. dev., n=3 biological replicates) B. The kinetics of endocytosis was determined by pulse-chase uptake of the pH-sensitive probe TL:FITC. Cell-associated fluorescence of live cells was measured over time by flow cytometry. Data are normalized to initial bound ligand and are presented as MFI (mean ± std. dev., n=3 biological replicates). Inset shows expanded graph at the earliest time points. C. Cells were pulsed with TL:Bio (30 min, 37°C), washed, and then reincubated (20 min, 37°C) to chase the lectin probe into terminal endocytic compartments. Imaging was performed on fixed/permeabilized cells stained with anti-p67 (red) and streptavidin:A488 (green). Representative 3-channel images are presented of control (tet−) and silenced cells (tet+). Inserts are corresponding single channel images of the lysosomal region. Cell outlines were captured from matched DIC images. Bar indicates 2 μm.