Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor

Figure 4

Effect of PAF exposure (pre-fMLF or post-fMLF) on relative FPR1 recognition by NFPR mAbs. Twenty-four wells of a 96-well microtiter plate were loaded with 5.0 × 10⁵ PMN in twelve well sets. The cells were exposed to 50 μL dilutions of PAF ranging from 0 to 100 nM in RPMI and incubated at 37°C for 10 min, followed by addition of 5 μL of 5 μM fMLF (+) or control RPMI buffer and then incubated for an additional 10 min (upper graph labeled [PAF]:pre-fMLF). Alternatively, the order of additions was reversed except that incubation with PAF was for 5 min ([PAF]: post-fMLF). The supernatants were then removed; the cells extracted with TL buffer at 4°C and processed as normally as described in the legend of Figure 3 and Materials and Methods. The 60 K-FPR1 was then quantitated utilizing LI-COR Odyssey Image Studio software. The paired NFPRb/NFPRa integrated intensity ratios were calculated (see Supplement to Figure 4) and plotted as a function of the log of PAF concentration with the zero concentration plotted on the left ordinate. Error bars represent SEM for four measurements using cells from two blood donors. A representative immunoblot and Odyssey quantitation method are shown in Figure 4 Supplement.