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

Figure 3

Degree of FPR1 proteolysis depends on conditions of cell solubilization. Six wells on each of two 96-well microtiter plates were loaded with 5 × 10⁵ PMN in 50 μL RPMI buffer and exposed to 1 μM fMLF (+) or RMPI vehicle and equilibrated for 10 min at 37°C as described in Section 2. Subsequently, the supernatants were removed and replaced with 50 μL of ice-cold DPBS. One plate was placed on ice and the other kept at room temperature for 2-3 min followed by careful replacement of the added buffer with ice-cold TL buffer or room temperature TS buffer. The plates were agitated at 4°C and room temperature for 10 min, respectively, and reduced and alkylated normally as described in Section 2. Two identical sets of 12 lanes were loaded with alternating lanes containing (13.3 × 10⁴ ceq/lane) extracts of cells exposed to 1 μM fMLF (+) or control buffer (−) in sets of six. Those that had been extracted at room temperature with TS buffer are denoted with a dotted line and SDS label above. Those extracted at 4°C with TL buffer are denoted with a solid line with LDS label above it. The blotted transfer was cut into two identical halves, incubated with NFPRa (left half) or NFPRb (right half), and developed as described above. The broad band representing FPR1 is identified with a vertical black bar and FPR1 label between the two blots. The position of FPR2 detected by NFPRa is also similarly shown. The 25K-FPR1() fragments are identified with diagonal arrows beginning at either 25K-FPR1 on the NFPRa blot and 25K-FPR1 on the NFPRb blot. The positions of the relative molecular weight markers, Mr, are shown to the left of each blot with horizontal dashes. From top to bottom their values are 125, 78, 41ǂ, and 27 thousand. Each experiment was performed in triplicate or duplicate from two separate PMN donors. (ǂ: omitted for clarity on the NFPRb blot).