(A) Effect of sphingoid bases on ERK1 and ERK2 activation during fMLP-primed phagocytosis of EIgG in PMN. PMN (2 × 106/mL) were incubated with different sphingoid bases (5 and 10 μmol/L), buffer (control), or 50 μmol/L PD098059 for 30 minutes at 22°C. The PMN were primed with fMLP (10−7mol/L) for 10 minutes at 37°C, followed by the addition of EIgG (1 × 108/mL) for 3 minutes at 37°C. The membranes were probed with anti-MAP kinase Ab that recognizes both phosphorylated isoforms, ERK1 (p44) and ERK2 (p42). (B) Effect of sphingosine on ERK1 and ERK2 activation. PMN (2 × 106/mL) were preincubated with 10 μmol/L sphingosine or buffer (control) and subsequently activated with 10−7 mol/L fMLP and EIgG. Phagocytosis was allowed to proceed for 3 minutes. The samples were run on 10% SDS-PAGE and protein transferred to PVDF membranes. The membranes were probed with Ab against phosphorylated ERK1 and ERK2. The figure is representative of three experiments. (C) Kinetics of ERK1 and ERK2 phosphorylation in PMN during phagocytosis of EIgG. PMN were incubated with EIgG. At the indicated times, phagocytosis was terminated and the PMN were treated as noted in (B).
