Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1 - Effect of μ2- or α-siRNA treatment on the cell surface levels of Lamps. HeLa cells were transfected twice with siRNAs directed to the μ2 or α subunits of AP-2. Forty-eight hours after the second round of transfection, the cell surface levels of TfR, MHC-I, and Lamps in mock- (thin open curve), μ2-siRNA- (bold open curve) or α-siRNA-treated cells (grey filled curve) were quantified by FACS analysis.
• Supplemental Figure 2 - Effect of clathrin or AP-2 depletion on the cell surface levels of Lamps in Mel JuSo cells. (A) Mel JuSo cells were transfected twice with siRNAs directed to CHC or the μ2 subunit of AP-2. Forty-eight hours after the second round of transfection, equal amounts of homogenates of mock- and siRNA-treated cells were subjected to SDS-PAGE and immunoblotting using antibodies to μ2 or CHC. (B) The cell surface levels of TfR and Lamps in mock- (thin open curve), CHC-siRNA- (bold open curve) or μ2-siRNA-treated cells (grey filled curve) were quantified by FACS analysis.
• Supplemental Figure 3 - Inhibition of TfR and CD63 internalization caused by depletion of AP-2. Mock- and μ2-siRNA-treated HeLa cells were incubated with antibodies to TfR (A) or CD63 (B) for 1 h at 4°C. After washing off the unbound antibodies, cells were incubated at 37°C for the indicated periods. Cells were then incubated with a PE-conjugated anti-IgG and analyzed by FACS. Values for the internalization of CD63 are the mean ± SD from three independent experiments.
• Supplemental Figure 4 - Distribution of TfR and Lamps in mock-, μ1A-, μ3A-, and μ4-siRNA-treated, permeabilized HeLa cells, analyzed by indirect immunofluorescence and confocal microscopy. Bar, 5 μm.
• Supplemental Figure 5 - Comparison of the localization of CD63 and internalized Tf in mock-, μ1A-, μ3A-, and μ4-siRNA-treated cells. Live HeLa cells were incubated for 25 min at 37°C with 10 μg/ml Alexa 488-conjugated Tf (green) and then were fixed, permeabilized and stained for CD63 (red) and analyzed by confocal microscopy. Merged images are shown in the third column. Bar, 5 μm.
• Supplemental Figure 6 - Comparison of the localization of Lamp-1 and internalized Tf or TGN upon combined depletion of AP complexes. (A). Live HeLa cells were pulsed for 25 min with 10μg/ml of Alexa 488-conjugated transferrin (green) and then fixed, permeabilized and stained for Lamp-1 (red) and analyzed by confocal microscopy. (B) Transfected Hela cells were fixed, permeabilized and co-stained for Lamp-1 (red) and TGN46 (green), and analyzed by confocal microscopy. Bar, 5 μm.