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Fig. 6. tTG is colocalized with LRP1 during internalization and the two interact on the cell surface. (A) tTG colocalizes with LRP1 early after endocytosis from the cell surface. Antibody-uptake experiments were performed for 5 or 15 minutes with WI-38 fibroblasts and the Fab fragments of mouse mAb 4G3 against tTG and rabbit antibody Rb2629 against LRP1, as described above in Fig. 1A. The internalized tTG–4G3-Fab and LRP1–anti-LRP1 complexes were detected by immunofluorescence after cell permeabilization. Note the significant colocalization of the internalized proteins in the numerous peripheral endocytic vesicles. (B) tTG is associated with LRP1. Extracts of quiescent (–) and PDGF-treated (+) NIH3T3 cells expressing exogenous tTG were subjected to immunoprecipitation with non-immune IgG or antibody 2629 against LRP1. The resulting immune complexes were examined by SDS-PAGE and immunoblotting for tTG and LRP1. (C) tTG enhances the association of LRP1 with β1 integrins. β1 integrins were immunoprecipitated from the extracts of quiescent (–) or PDGF-treated (+) NIH3T3 cells lacking (vector) or expressing tTG. The resulting immune complexes were analyzed for LRP1, tTG and β1 integrins by SDS-PAGE and immunoblotting. (D) Cell-surface tTG mediates a shift of LRP1 towards high-density membrane or ECM fractions enriched in tTG and fibronectin. NIH3T3 fibroblasts lacking (vector) or expressing tTG were surface-biotinylated with membrane-impermeable sulfo-NHS-LC-biotin. Carbonate extracts at pH 11 of the cells were subjected to ultracentrifugation in a discontinuous (45%-35%-5%) sucrose gradient. Biotinylated (cell surface) proteins from each gradient fraction were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against LRP1, tTG and fibronectin. The high-density fractions enriched in tTG and fibronectin and expressing increased amounts of LRP1 in the tTG-expressing cells are marked with asterisks. The numbers beneath the LRP1 bands show relative intensities compared with the value of 1.0 assigned to cells lacking tTG in the absence of PDGF. Shown are representative results of three independent experiments.
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