Finding the triggers of inflammation Hudson_Research NatureComms
BMDMs were passaged in DMEM containing 10% fetal bovine serum , 50 U/ml penicillin and 50 μg/ml streptomycin and supplemented with 15–20% L929 cell conditioned medium. Cells were infected with retrovirus containing murinecloned into pMIGMCS . Macrophages were subsequently sorted by flow cytometry for GFP expression and analyzed as indicated in the figure legends.
was transfected using Lipofectamine 2000. After 48 h, cells were washed with ice-cold PBS and lysed with 700 μl of non-denaturing DISC lysis buffer , 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100, Roche complete protease inhibitor cocktail, 10 μM PR619 and 1 mM NEM) on ice for 30 min. Cell lysates were spun at ~13,000× and the supernatant collected. A 50 μl slurry of M2 agarose anti-FLAG conjugated beads was added to the clarified cell lysate and rotated overnight at 4 °C.
eluted three times with 50 μl of elution buffer at 65 °C. Eluted proteins were run on NuPAGE Novex 4-12% Bis-Tris gel . Ruby staining was used to view the purified proteins, in-gel trypsin digestion performed, and digested peptides analyzed by high resolution mass spectrometry, as previously described
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