Supplemental Material

This article contains the following supporting material:

• Figure 1 - Depletion of Tsg101 and Hrs by second siRNA. A-B
  A431 cells were nucleofected with the siRNAs against Tsg101 or Hrs which were used for all the experiments shown in Figs. 1-10 (Si1) or a second siRNA which targets a different region of either the Hrs or Tsg101 RNA (Si2).

  A shows the efficiency of depletion using siRNAs against Tsg101 and Hrs by western blot.
  B shows confocal images of siRNA treated cells after 2h stimulation with Alexa 488-EGF. Note that in both Tsg101 siRNA treated cells EGF is present in tubular clusters. Both siRNAs directed against Hrs caused the enlargement of endosomes. Bars, 10μm.

• Figure 2 - Depletion of Tsg101 enhances TF recycling.
  A-B, Control (Inv) and Tsg101 siRNA-treated A431 cells were incubated with ¹²⁵I-TF for 30 minutes at 37°C. TF was removed and cells were surface stripped and incubated in fresh pre-warmed binding medium. After each time point the medium was collected and replaced by fresh medium for the subsequent time point. The data presented excludes the stimulation period. Data represents 3 separate experiments.

  A shows enhancement of TF recycling at different time points using TF free chase medium.
  B shows the kinetics of 125I-TF recycling at the 1h time point without and with excess unlabelled TF. TF recycling was increased in Tsg101-depleted cells whether or not the cold TF was present in the chase medium.

• Figure 3 - Distribution of MVBs within the cytoplasm in control and Hrs-deleted cells.
  Control (Inv) and Hrs-depleted (Hrs) A431 cells were incubated with anti-EGFR gold-conjugated antibody and EGF for 2h at 37°C. Cells were fixed and prepared for TEM. Micrographs show thin (70nm) sections. The letters N, L, C and M represent nucleus, lysosome, centriole and MVB respectively.