Integrin ligation up-regulates the turnover of TSP-1 via CD47. (A) Comparison of the turnover of TSP-1 (Ab4) in PBTs on different substrata and in suspension (91% of the adherent cells on PLL and fibronectin were CD3⁺). The cells were incubated for 30 minutes in the absence or presence of brefeldin A (10 μg/mL). (B) 4N1K, a peptide corresponding to the C-terminal CD47-binding domain of TSP-1, at a concentration of 50 μM inhibits the turnover of cell-surface TSP-1 in T cells on fibronectin (10 μg/mL) and ICAM-1 (2 μg/mL), whereas a scrambled control peptide, Sc4N1K (50 μM), does not affect TSP turnover. Lymphocytes were allowed to adhere to fibronectin for 30 minutes in the presence or absence of peptides before fixation (94% of the PBTs were CD3⁺). (C) 4N1K augments cell-surface TSP-1 and reduces intracellular TSP-1, indicating that binding to CD47 induces internalization of cell-surface TSP-1. AF24 cells were allowed to adhere to glass slides coated with fibronectin in the absence or presence of 4N1K or control peptide for 15 minutes before fixation. TSP-1 expression was measured using quantitative immunocytochemistry. One representative experiment of 3 to 4 independent experiments is shown. Error bars: Statistics: Mann-Whitney U test (C).