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

Figure 2

25K-FPR1() is insensitive to modulating physiologic conditions. (a) The 25K-FPR1() bands persist when adherent PMNs are depleted of ATP. Adherent PMN were incubated with DPBS that contained 40 mM NaF, replacing 40 mM NaCl in the composition of DPBS buffer, prior to treatment with 1 μM fMLF or vehicle for 10 min as described in the legend of Figure 1. The 25K-FPR1 or 25K-FPR1 bands in line with the Mr 25 kDa are boxed for clarity. The treatment of the cells with or without NaF (+/−) or fMLF (+/−) are so marked in the upper two rows adjacent to their respective labels. The first two lanes, marked “S” adjacent to the 25 K marker (boxed left), show no density in either NaF-treated (+) or untreated (−) PMN indicating that no Mr = 25K bands are being generated by suspension PMN when they are fMLF-treated (+) or untreated (−). The center boxed 4 lanes show the density of the 25 K band in adherent cells for NFPRa (upper) and NFPRb (lower). One of three experiments. (b) The 25K-FPR1 persists unaffected when PMN are allowed to adhere at low temperature. Adherent PMN were not centrifuged but allowed to adhere by settling for 0 to 20 min either at 20°C (lower) or 4°C (upper) before extraction by TS buffer. The 25K-FPR1 was digitally excised from the NFPRb immunoblot scan. The density of the 4°C 25K-FPR1 band appears to be higher than that of the 25K band generated after the 25°C settling assay because they represent different plates incubated at different temperatures. Plate to plate variation is greater than well to well variation. One of two experiments. (c) The 25K-FPR1 persists when PMN are allowed to adhere onto different coatings. Wells of 96-well microtiter plates were incubated with 100 μL of 1 mg/ml autologous human IgG, ConA, succinyl ConA (SConA), wheat germ agglutinin (WGA), and protein-free DPBS. The 25K-FPR1 band was digitally excised from the NFPRb immunoblot scans and is displayed showing the density of the 25K-FPR1 from adherent cells stimulated (+) and unstimulated (−) with 1 μM fMLF. Scanned blots showing different coatings were derived from different experiments on different days and so are not correlated in density with one another. One of four experiments.

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