Complete ultra protease inhibitor cocktail

Total protein was extracted from frozen half-brains by homogenization in cold lysis buffer (20 mM Tris, 0.25 M sucrose, 2 mM EDTA, 10 mM EGTA, 1% Triton X-100 plus 1 tablet of Complete ULTRA protease inhibitor cocktail [Sigma, St. Louis, MO] per 10 mL solution). Homogenates were centrifuged at 100,000g for 40 min, supernatant was collected, and protein levels determined using the BCA protein assay reagent kit (PIERCE, Milwaukee, WI). Levels of TNFα, Ccl2, IL-1ra, and IL-4 were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN), as per the manufacturer’s instructions.

GFP pull-down samples were purified using from Phoenix-ampho cell line transiently transfected (16 h) with 6 µg of the indicated plasmids in a 6 cm petri dish using FuGENE VI as described above. Cells for each experiment were lysed in 500 µl RIPA supplemented with cOmplete™ ULTRA protease inhibitor cocktail (Sigma Cat#5892988001) and 5 µM M344 (Stratech Scientific Cat#S2779-SEL) on ice for 30 min and subjected to mechanical disruption using 25 gauge needle till no visible clump can be seen. 100 µl of washed, pre-equilibrated GFP-trap (chromotek Cat#gtma-100) in lysis buffer was added to each precleared (14,000 × g for 10 min) sample, incubated overnight, 4 °C, washed thrice in RIPA without NP-40 and eluted in LDS, 95 °C for 10 min before SDS-PAGE.