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Fig. 3. Association between TfR2 and caveolin-1 in LDTI membranes as shown by immunofluorescence and immunoprecipitation experiments. (A) HepG2 cells were plated on coverslips and left to adhere overnight. Cells were stained with a mouse mAb directed against TfR2, followed by a Texas-Red-conjugated goat anti-mouse IgG. Cells were then fixed with paraformaldehyde (PFA, top panels) or with methanol/acetone (bottom panels), permeabilized and stained with a rabbit polyclonal anti-caveolin-1, followed by a FITC-conjugated anti-rabbit IgG. (B) HepG2 cells were stained with anti-TfR2 (red), then fixed (PFA) and labeled with a FITC-conjugated anti-CD81 mAb. (C) HepG2 cells were labeled with anti-TfR1 mAb, followed by a Texas-Red-conjugated goat anti-mouse IgG. Cells were then fixed and permeabilized (methanol/acetone, saponin) and stained with a rabbit polyclonal anti-caveolin-1, followed by a FITC-conjugated anti-rabbit IgG. (D) HepG2 cells were stained using the indicated primary mAbs, followed by a FITC-conjugated goat anti-mouse IgG. Where indicated, stained cells were exposed to Triton X-100 (TX-100) before analysis. Nuclei were counterstained using Syto59. (E) LDTI domains prepared from biotinylated HepG2 cells were immunoprecipitated with anti-TfR2 mAb. Top panel shows detection of immunoprecipitated (IP) and total (TOT) LDTI by anti-TfR2 or by streptavidin-horseradish peroxidase conjugated (strept). Bottom panel shows detection of IP and total LDTI by anti-caveolin 1 antibody. Only externally exposed biotinylated proteins were detected by streptavidin-HRP. As a control, an irrelevant antibody was used for immunoprecipitation (IgG). Positions of molecular size markers in kDa are indicated on the left. (F) Whole cell extracts prepared from biotinylated HepG2 cells were immunoprecipitated with a mouse mAb directed against TfR1. Total lysate (TOT) and immunoprecipitated fraction (IP) were detected by streptavidin-HRP-conjugated (strept) anti-TfR1 (top panel) and by anti-caveolin-1 (bottom panel). As a control, an irrelevant antibody was used for immunoprecipitation (IgG).
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