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Western blotting analysis. To detect beta interleukin 1 (163-171) recombinant NR proteins, the purified microsomal fraction from the fat bodies or the baculovirus fraction from the body fluid was fractionated by SDS–PAGE and transferred to PVDF membrane. The membrane was incubated with 1/1000 diluted primary antibody at 4 °C overnight and incubated with 1/25,000 diluted HRP-conjugated anti-mouse IgG antibody. The positive bands were visualized using the ECL-Plus (GE Healthcare).
[35S]GTPγS-binding assay. [35S]GTPγS (1250 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA). The membrane preparation expressing Giα-fused NRs (20 μg of total protein) was incubated in 100 μl of 20 mM HEPES–KOH (pH 8.0), containing 1 mM EDTA, 160 mM NaCl, 1 mM DTT, 100 pM [35S]GTPγS, 1 μM GDP, and 10 mM MgCl2, at 30 °C for 30 min in 96-well microplates with or without nociceptin peptide. [35S]GTPγS bound to the membrane was trapped on a bolus GF/B glass fiber filter (Whatman, London, UK). The GF/B filter was washed three times, and then the radioactivity was counted with liquid scintillation counter (LS6500, Beckman Coulter, Fullerton, CA).