Enhanced chemi luminescence (ecl)

Cell pellets were resuspended in RIPA buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM NaF, 1 mM Na orthovanadate, 2.5 mM Na pyrophosphate, 2 mM β-glycerophosphate) supplemented with protease inhibitor tables (Roche) followed by two 30-sec rounds of sonication (65% amplitude) using a cup horn (Misonex 3000). After clarification of the lysates by centrifugation, protein concentrations were determined using the BCA assay kit (Pierce). Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were then incubated with the appropriate primary and secondary antibodies. Signal detection was performed with ECL (Genesee Scientific). Antibodies are listed in Supplemental Table S2.

BMDMs (3 × 10⁶ per well in 6-well cell culture plates) or RAW264.7 cells (2 × 10⁶ per well in 6-well cell culture plates) were treated with LPS 10 or 100 ng/ml for 30 min and lysed in RIPA buffer with protease and phosphatase inhibitors (Roche, Nutley, NJ). Western blot analysis was performed with primary antibodies against p-CREB-S133, CREB, IRS1, IRS2, p-AKT-S473, AKT, p-AKT1-S473, AKT1, p-AKT2-S474, AKT2, p-NF-κB P65-S536, NF-κB P65, IκBα, β-actin, and GAPDH from Cell Signaling Technology (Denvers, MA) and GHSR from Invitrogen (Waltham, MA). Signaling was visualized with ECL (Genesee Scientific, San Diego, CA) and analyzed using Image J software.