JCS020404 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. The PI(3) kinase inhibitor LY294002 does not block ErbB2 internalization. SKBr3 cells expressing EGFP-2×FYVE were left untreated (A) or treated with 100 µM LY294002 for 2 hours (B) before binding anti-ErbB2 antibodies on ice and warming to allow internalization for 5 minutes. After acid-stripping, cells were fixed, permeabilized and stained with Texas-Red goat anti-mouse IgG antibodies. Scale bar: 10 µm. Similar amounts of internalized ErbB2 were detected with and without LY294002 treatment. In control cells, EGFP-2×FYVE fluorescence was visible on endosomes (A) as well as in the cytosol, where it was especially evident at high expression levels. In LY294002-treated cells, EGFP-2×FYVE labeling of small punctae characteristic of early endosomes was diminished, consistent with a loss of PI(3)P from these structures (B). However, in many cells, especially at high expression levels, enlarged structures showing intense EGFP-2×FYVE fluorescence were still detectable after drug treatment (A.G.O.-F., unpublished). This may have resulted from shielding of endosomal PI(3)P from phosphatases by high amounts of bound EGFP-2×FYVE.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Newly internalized ErbB2 colocalizes with CtxB, PLAP and dextran in serum-starved cells. Cells were serum-starved overnight, pretreated with GA for 1 hour, subjected to antibody or toxin binding as appropriate, warmed for 2 minutes, acid-stripped, fixed and permeabilized. Internalized anti-ErbB2 antibodies were then detected with appropriate secondary antibodies. (A) Anti-ErbB2 antibodies and AF-594-CtxB (0.5 µg/ml) were bound to cells. (B) Fl-anti-PLAP Fab fragments and anti-ErbB2 antibodies were bound to cells. (C) Anti-ErbB2 antibodies were bound to cells, which were warmed with 1 mg/ml FluoroRuby dextran. Epifluorescence images are shown. ErbB2, left; CtxB, PLAP or dextran, middle; merged images, right. Scale bar: 10 µm.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Dominant-negative forms of Cdc42 and RhoA do not block ErbB2 endocytosis. Anti-ErbB2 antibodies were bound to SK-BR-3 cells transfected with wild-type (not shown) or dominant-negative forms of 3×HA-RhoA (A) or 3×HA-Cdc42 (B) on ice. Cells were warmed for 5 minutes, subjected to acid stripping, fixed and permeabilized before immunofluorescent detection of dominant-negative 3×HA-RhoA or 3×HA-Cdc42 (red), together with internalized anti-ErbB2 (green). (C) At least 100 transfected cells on each slide were counted and scored for internalization of ErbB2 (at least three punctae/cell). Results shown are the average of two experiments. The range is shown.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. ErbB2 accumulates in late endosomes and lysosomes in chloroquine-treated SK-BR-3 cells. SK-BR-3 cells were treated with GA and 100 µM chloroquine for 5 hours, fixed, and permeabilized. Left panels: ErbB2 was detected with polyclonal (A) or monoclonal (B) antibodies and appropriate secondary antibodies. Middle panels: (A) endogenous CD63; (B) endogenous LAMP1. Right panels: merged images. Epifluorescence images are shown. Scale bar: 5 µm.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. ErbB2 internalization and trafficking in GA-treated COS-7 cells. COS-7 cells were transiently transfected with ErbB2, together with Thy1.1 or dominant-negative HA-dynamin where indicated. (A) After 1 hour GA treatment, anti-ErbB2 antibodies (labeled with the Zenon AF-488 labeling kit when visualizing ErbB2 together with Thy1 or FluoroRuby dextran) were bound to COS-7 cells on ice, together with AF-594-CtxB (0.5 µg/ml) or AF-594-anti-Thy1 Fab fragments where indicated. Cells were warmed for 2 minutes, together with AF-594-Tf or FluoroRuby dextran (1 mg/ml) where indicated, acid-washed and processed for IF, permeabilizing cells and detecting internalized anti-ErbB2 antibodies with secondary antibodies only when the Zenon labeling kit was not used. (B) Cells co-transfected with ErbB2 and dominant-negative HA-dynamin were treated with GA for 1 hour before binding anti-ErbB2 antibodies and warming for 5 minutes with AF-594-Tf. After acid washing, cells were processed for IF, detecting internalized anti-ErbB2 antibodies with secondary antibodies and HA-dynamin with rabbit anti-HA and AF-350 goat anti-rabbit antibodies. (C) ErbB2-transfected cells were treated with GA for 3 hours (top panels) or 5 hours (bottom panels). Cells were processed for IF, detecting ErbB2 together with EEA1 (top) or CD63 (bottom) by indirect IF. Scale bar: 10 µm.