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Figure 4


Fig. 4. Cell-surface tTG is internalized by means of clathrin- and lipid-raft-(caveolae-) dependent pathways. (A) Cell-surface tTG colocalizes with transferrin and clathrin during internalization. Antibody-uptake assays were performed with WI-38 fibroblasts as described above in Fig. 1A with the Fab fragments of mouse mAb 4G3 against tTG and FITC-labeled transferrin (upper panels), or the anti-tTG Fab fragments only, and the cells were co-stained for clathrin after 15 minutes of internalization (lower panels). (B) Internalization of cell-surface tTG is attenuated by the inhibitors of clathrin- and caveolae-dependent endocytosis. WI-38 fibroblasts were left (c) untreated or were treated with (h) hyperosmotic 0.45 M sucrose or (f) 5 µg/ml filipin before surface biotinylation with membrane-impermeable SH-cleavable sulfo-NHS-SS-biotin. (C) Co-distribution of tTG with caveolin-1 and beta1 integrins on the cell surface. Live non-permeabilized MRC-5 fibroblasts were triple-labeled with antibodies against the three proteins. Arrows mark the sites of colocalization of tTG and caveolin-1 on the cell surface. (D) Inhibition of caveolae-dependent endocytosis increases cell-surface levels of tTG. NIH3T3 fibroblasts expressing tTG were either left untreated or were treated with filipin. Surface labeling of untreated and filipin-treated cells with sulfo-NHS-LC-biotin was followed by isolation of biotinylated (cell surface) tTG. (E) Downregulation of caveolin-1 promotes endocytosis of cell-surface tTG. Surface biotinylation and endocytosis assays were performed with U-251 glioma cells expressing vector only or caveolin-1 siRNA, using membrane-impermeable SH-cleavable sulfo-NHS-SS-biotin. Biotinylated proteins in (B,E) were isolated and tTG was detected by SDS-PAGE and immunoblotting in the fraction of proteins internalized from the cell surface. The numbers beneath the tTG bands show relative intensities compared with the value of 1.0 assigned to untreated cells (D) or cells before internalization (B,E). Shown in (B,D,E) are representative results from three independent experiments.