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Supporting Information

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Appendix S1: Materials and methods.

Figure S1: Increased amounts of Tsg101 are membrane associated and sorted into ILVs upon expression of Rab5(Q79L), and the localization of Tsg101 in the MVE lumen is detected by trypsin protection in various cell lines. A) Cytosol and membrane fractions were isolated from normal HEp2 or HEp2-Rab5(Q79L) cells. Equal amounts of membrane fractions (35 µg) were treated without (lanes 2 and 5) or with (lanes 3 and 6) 4 ng proteinase K/µg membrane fraction to determine whether the relative amount of Tsg101 sorted into ILVs is affected by the expression of Rab5(Q79L) giving rise to the enlarged endosomes. The experiment was repeated twice and a representative blot is shown. B) Cytosol and membrane fractions were isolated from HEp2 cells and equal amounts of protein (35 µg) were subjected to SDS–PAGE. The membrane fraction was digested (or not) with increasing concentrations of an alternative protease, trypsin, for 15 min at 37°C. The samples were separated by SDS–PAGE and immunodetectable levels of Tsg101, Hrs, Lamp1 and tubulin were assessed. C) Equal amounts of cytosol and membrane proteins (35 µg), the latter treated (or not) with 4 ng proteinase K/µg membrane protein, were isolated from PC-3 or U2OS cells. Immunoreactive Tsg101 was detected by western blotting analysis. The purity of the cytosol and membrane fractions was verified with antibodies against the specific markers, tubulin and Lamp1. The experiments were performed at least twice, and representative results are shown.

Figure S2: Decreased amounts of Tsg101 are protease protected in cells depleted of Hrs and SNX3. Hep2 cells were transfected with siRNA against Hrs and SNX3, and split the next day. Three days after transfection, cytosol and membrane fractions were isolated and equal amounts of membrane fractions (35 µg) were treated without (−) or with (+) 4 ng proteinase K/µg membrane fraction. This was done to determine the relative amount of Tsg101 protected in ILVs in cells depleted of Hrs or SNX3. The experiment was repeated at least three times and a representative blot is shown.

Figure S3: MVE biogenesis does not require serum stimulation of cells. A) HEp2-Rab5(Q79L) cells cultured in the presence and absence of serum were examined by electron microscopy. The amounts of ILVs were approximately equal in cells grown in 10% FCS and serum-starved cells (zoomed-in areas are shown in the right column). B) To determine the distribution of the MVE marker CD63 we labeled cells with a mouse anti-CD63 antibody, followed by a bridging secondary mouse anti-rabbit IgG and 10-nm protein A gold. We detected intraluminal CD63 labeling (arrowheads) in both control and serum-starved cells. Scale bars as indicated.

Figure S4: Knockdown of c-Cbl and Cbl-b inhibits ubiquitination and degradation of EGFR upon incubation with EGF. HEp2-Rab5(Q79L) cells transfected with siRNA against c-Cbl and Cbl-b, the day after transfection cells were spilt and experiment were performed on the fourth day. Induction of endosomes was induced by adding CdCl2 the day prior to the experiment. A) Cells were incubated with or without EGF (50 ng/mL) for 10 min before hot lysis and immunoprecipitation of EGFR under denaturating conditions. The immunoprecipiatated proteins were subjected for western blotting using antibodies against ubiquitin and EGFR. The experiment was done at least three times and a representative blot is shown. B) To measure the degradation of EGF, cells were incubated with 125I-EGF (50 ng/mL) at 37°C for 15 min, before cells were washed in PBS to remove excess of 125I-EGF and further chased at 37°C for the time indicated. The ratio of degraded, recycled and internalized 125I-EGF was calculated as described in Appendix S1 and plotted against time. The data represent the average of the three independent experiments (±SE).

Figure S5: Depletion of the ubiquitin ligase Mahogunin decreases the level of Tsg101 in ILVs of MVEs. HEp2-Rab5(Q79L) cells were transfected with control or Mahogunin siRNA for 5 days. A) Cells were lysed and subjected to western blotting with antibodies against Mahogunin and actin. One representative blot is shown. B) The day after the transfection, cells were split onto coverslips and enlarged endosomes were induced on the fourth day upon transfection by adding CdCl2. To determine the distribution of Tsg101 in enlarged endosomes, cells were labeled with Tsg101 and EEA1 antibodies. The graph represents quantifications of Tsg101 localization to enlarged endosomes in control versus Mahogunin siRNA cells. Images were acquired using the same setting by the Zeiss LSM 510 META confocal microscope and the graph represents the average of the three independent experiments (±SE).

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TRA_1220_sm_s1.doc31KSupporting info item
TRA_1220_sm_f1.jpg451KSupporting info item
TRA_1220_sm_f2.jpg500KSupporting info item
TRA_1220_sm_f3.jpg632KSupporting info item
TRA_1220_sm_f4.jpg237KSupporting info item
TRA_1220_sm_f5.jpg230KSupporting info item

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