(A) Immunoblot analysis of human PMN membrane proteins with the FLAP antiserum anti-H5. Membranes from 15×10⁶ PMN equivalent were resuspended in HBSS and treated with the cross-linker sulpho-HSAB (20 μg/ml) for 15 min. The reaction was stopped with sample buffer and proteins were analysed by SDS/PAGE using a 5–20% gradient. (B) Immunoblot analysis of Sf9 cell membrane proteins with the FLAP antiserum anti-H4. Sf9 cells were infected for 3–4 days with a baculovirus containing FLAP. Sf9 cells were sonicated in HBSS and membranes from 5×10⁶ cell equivalents were resuspended in HBSS. Cross-linking and electrophoresis were performed as in (A). (C) Gel-strip 2D-electrophoresis of Sf9 cell membranes. The sample was prepared as in (B), treated with the cross-linker and processed as described in the Material and methods section. Proteins were separated first on a pH 3–10 gradient strip and then by 5–20% SDS/PAGE gradient. The membrane was blotted with the FLAP antiserum anti-H5. M-H, membrane treated with the cross-linker sulpho-HSAB; M, untreated membranes. Results shown are from one experiment and are representative of four different experiments.

Sf9 cells were cultured and infected with baculoviruses. Post infection (3–4 days), Sf9 cells expressing the various FLAP mutants were sonicated and the lysates were treated with the cross-linking agent sulpho-HSAB (20 μg/ml) as described in the Materials and methods section. The reaction was stopped with the sample buffer and the proteins were analysed by SDS/PAGE using a 5–20% gradient and the FLAP antiserum anti-H5. Results shown are from one experiment and are representative of three different experiments.

(A) and (B) Sf9 cells were infected with 5-LO baculoviruses one day before infection with the FLAP mutant D62N baculoviruses to insure adequate expression of the 5-LO, as described in the Materials and methods section. After infection (3 days) with the FLAP mutant baculoviruses, cells were exposed to various concentrations of AA for 5 min, sonicated and the lysates were treated directly with the cross-linker DFDNB (5 μg/ml) for 15 min (membrane enrichment procedures strongly decreased the level of 5-LO complex). The reaction was stopped with sample buffer and the proteins were analysed by SDS/PAGE using a 5–20% gradient. (A) Shows immunoblot analysis using the 5-LO antibody. (B) Shows immunoblot analysis using the FLAP antiserum anti-H5. (C) Sf9 cells were infected with the baculovirus containing 5-LO, or with the baculovirus containing 5-LO, and increasing amounts of the baculovirus containing the FLAP mutant D62N. The presence of D62N, D62N-dimer, 5-LO and 5-LO-containing complex in the infected Sf9 cells was analysed as described above (A) and (B). The amount of baculoviruses used for Sf9 infection is expressed as μl of baculovirus suspension added to the Sf9 cells. The bands at approx. 80 and 150 kDa are Sf9 cell proteins (see Results section) and their detection in immunoblot analysis using the 5-LO antibody was highly variable (e.g. bands not seen in 5A). Results shown are from one experiment and are representative of three different experiments.

(A) Effect of AA on FLAP dimer level. Membranes from 15×10⁶ PMN equivalent were pre-treated with AA for 5 min before the addition of the cross-linker sulpho-HSAB (20 μg/ml), as described in the Materials and methods section. Total proteins were analysed by SDS/PAGE (10–20% gradient) and the membrane was blotted with the FLAP antiserum anti-H5. (B) Effect of AA on 5-LO translocation and 5-LO product biosynthesis. Intact PMN (5×10⁶) were pre-treated with adenosine deaminase (0.3 unit/ml) for 5 min at 37 °C and then stimulated with the indicated concentrations of AA for 5 min. Nuclei were obtained by 0.1% NP 40 hypotonic lysis as described in the Materials and methods section. Nuclear proteins were analysed by SDS/PAGE (5–20% gradient) and the membrane was blotted with the 5-LO antibody. For the determination of 5-LO product biosynthesis, the reactions were stopped with cold (0 °C) methanol/acetonitrile (50:50, v/v) containing internal standards. 5-LO products were measured by reverse phase HPLC. Each incubation was performed in triplicate. LTB₄ represents the sum of LTB₄ and its ω-oxidation products (6-trans- and 6-trans-12-epi-LTB₄ are not detectable under these experimental conditions). (C) Experiments were performed as described in (B) with the exception that PMN were exposed to 100 nM MK-0591 for 5 min before stimulation with AA. Results shown are from one experiment and are representative of three different experiments.

(A) and (B) membranes from 15×10⁶ PMN equivalent were exposed to MK-0591 or BAY-1005 for 5 min before treatment with sulpho-HSAB (20 μg/ml), as described in the Materials and methods section. The reaction was stopped with sample buffer and proteins were analysed by SDS/PAGE using a 5–20% gradient and the FLAP antiserum anti-H5. Membranes were exposed for 30 s and 4 min for quantification of FLAP and FLAP-dimer respectively. (C) Intact PMN (10×10⁶/ml) were pretreated with various concentrations of the FLAP inhibitor MK-0591 in the presence of adenosine deaminase (0.3 unit/ml) for 10 min at 37 °C and then stimulated with 5 μM AA for 5 min. The incubations were stopped by addition of 1 vol. of cold (0 °C) HBSS and then centrifuged (1000 g for 1 min, 4 °C). The supernatants were denatured by addition of 0.5 vol. of cold (0 °C) methanol/acetonitrile (50:50, v/v) containing internal standards and 5-LO products were analysed as described in the Materials and methods section. The cell pellets were processed for nuclear 5-LO analysis as described in Figure 2(B) (legend). 5-LO products represent the sum of LTB₄ and its ω-oxidation products and 5-HETE. Results shown are from one experiment and are representative of three different experiments.