Fig. 3. The internalized cell-surface tTG undergoes proteolytic degradation. (A) tTG colocalizes with its binding partner, the β1 integrin subunit, early after endocytosis from the cell surface. Antibody-uptake experiments were performed for 15 minutes, as described above in Fig. 1A, with WI-38 fibroblasts and the Fab fragments of mouse mAb 4G3 against tTG and rat mAb 9EG7 against β1 integrins. (B) Cell-surface tTG, but not the β1 integrin subunit, is degraded after internalization. Surface biotinylation and endocytosis assays were performed with CHO cells and membrane-impermeable SH-cleavable sulfo-NHS-SS-biotin. Biotinylated proteins were isolated and tTG and β1 integrin were detected by SDS-PAGE and immunoblotting in the fraction of proteins internalized from the cell surface. The intensities of protein bands at various time points of internalization were quantified by densitometry and compared with those of cell-surface tTG and β1 integrins before endocytosis. Shown are the means ± s.d. for three independent experiments. Asterisks indicate proteolytic fragments of tTG.