BMDM were seeded at 106/ml in 6 well plates. The following day medium was replaced and cells were stimulated with 10 ng/ml LPS for 3 h. Medium was removed and replaced with SFM containing the indicated inhibitors or controls for 30 min, followed by the addition of 10 µM nigericin for 1 h. The supernatants were removed, cells were rinsed in ice-cold PBS and 500 µl ice-cold buffer (20 mM Hepes-KOH, pH 7.5, 150 mM KCL, 1%NP-40 0.1 mM PMSF, 1 mg/ml leupeptin, 11.5 mg/ml aprotinin and 1 mM sodium orthovanadate) was added. Cells were scraped and lysed by shearing 10 ィ through a 21 gauge needle. 50 µl of lysate was removed for Western blot analysis. Lysates were centrifuged at 330 × g for 10 min at 4 °C. The pellets were washed twice in 1 ml ice-cold PBS and resuspended in 500 µl PBS. 2 mM disuccinimydyl suberate (from a fresh 100 mM stock prepared from DSS equilibrated to RT and made up in dry DMSO) was added to the re-suspended pellets, which were incubated at RT for 30 min with rotation. Samples were then centrifuged at 330 × g for 10 min at 4 °C. The supernatant was removed and the cross-linked pellets were resuspended in 30 µl Laemmli sample buffer. Samples were boiled for 5 min at 99 °C and analysed by Western blotting.