Figure 4.

Chymotrypsin-induced loss of L-selectin reduced ANXA1 interaction with L-selectin. (a) Effect of different concentrations of chymotrypsin on U-937 expression of L-selectin. Cells were incubated with different doses of chymotrypsin (U (1 × 10⁶)⁻¹ cells) for 15 min at 37°C. The expression of L-selectin antigen was analyzed by flow cytometry using the FITC-DREG-56 antibody. Chymotrypsin induced a dose-dependent decrease in L-selectin expression with a maximal effect achieved at 10 U per 1 × 10⁶ cells (−80%). Bars indicate mean±s.e.m. of three independent experiments, (b) Loss of L-selectin antigen by chymotrypsin is associated with a decrease in the interaction between ANXA1 and L-selectin. U-937 cells (C) were stimulated with 1 nM PMA (P) for 24 h and then with 10 U per 1 × 10⁶ cells of chymotrypsin for 15 min at 37°C, before performing the immunoprecipitation with a specific ANXA1 antibody, as described above. L-selectin immunoreactivity was detected with DREG-56 (WB). Data shown are representative of three independent experiments giving similar results.