Supporting Information

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Figure S1: Rab35 depletion alters the localization of internalized transferrin without affecting transport kinetics. HeLa cells grown on coverslips were either mock treated or treated with Rab35-siRNA oligonucleotides for 72 h. A–G) Cells were incubated in DMEM containing 0.5% BSA (starvation media) for 30 min before adding transferrin-568 (Tf-568) for 5 min at 37°C. Cells were either fixed directly (A and B) or ‘chased’ in DMEM containing serum (complete media) for the indicated time-points (C–F). G) Quantification of Tf-568 uptake and recycling by flow cytometry. Bar, 10 µm.

Figure S2: Depletion of Rab35 does not affect β1 integrin uptake and recycling. HeLa cells were either mock treated or treated with Rab35-siRNA oligonucleotides for 72 h. Cells were then incubated in starvation media for 1 h prior to incubation with anti-human β1 integrin antibody for 1 h at 37°C. Cells were briefly acid stripped and either directly fixed (A and B) or chased in complete media for 2 h at 37°C before another round of brief acid strip and fixation (C and D). E) Quantification of β1 integrin uptake and recycling experiments. One hundred cells of each treatment from three independent experiments were quantified for their internalized β1 integrin mean intensity. Bar, 10 µm.

Figure S3: Depletion of Rab35 from Jurkat T cells causes enhanced levels of CD3 at the plasma membrane. Jurkat cells were treated for 48 h with Rab35 siRNA oligonucleotides. A) Lysates from mock- and Rab35-siRNA-treated Jurkat cells were immunoblotted with antibodies aganst actin (control; top panel) or Rab35 (bottom panel). B) Jurkat cells depleted of endogenous Rab35 were subjected to flow cytometry analysis with anti-CD3 antibodies. Four independent experiments were done using at least 10 000 cells each time. The p value was derived from a two-tailed Student's t-test for independent samples.

Figure S4: Efficient knockdown of Rab10 by siRNA. HeLa cells were either mock treated or treated with Rab10-siRNA for 72 h. In the last 24 h, both mock- and Rab10-siRNA-treated cells were transfected with HA-Rab10. Lysates from mock- and Rab10-siRNA-treated cells were separated by SDS–PAGE and immunoblotted with anti-actin (control) or anti-HA antibodies.

Figure S5: Over-expression of FLAG-EFA6 disrupts WT cherry-Rab8a localization to tubular endosomes. HeLa cells were transfected either with WT cherry-Rab8a (A), FLAG-EFA6 (B) or WT cherry-Rab8a and FLAG-EFA6 (C–E) for 24 h. Cells were fixed and immunostained with anti-FLAG antibody. Bar, 10 µm.

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