They were next lysed in buffer that contained 100 mM NaCl, 30 mM Tris-HCl (pH 8.5), 10% glycerol, 10 mM β-mercaptoethanol (2-ME), 5 mM imidazole, 1 mg ml−1 lysozyme and a mixture of protease inhibitors.
Cells were solubilized in lysis buffer containing 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate, 2 mM sodium orthovanadate, 1 mM sodium fluoride and mixture of protease inhibitors.
ARC and PVN were excised, and lysed in RIPA buffer containing a mixture of protease and phosphatase inhibitors (Sigma-Aldrich, St Louis, MO) supplemented with 10 mM Nicotinamide, 10 µM Trichostatin A, and 10 µM EX527.
Cells were lysed in lysozyme lysis buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 100 mM NaCl, 1% Triton X-100, 200 mM NaSCN, 1 mg/ml lysozyme) supplemented with a mixture of protease inhibitors (CompleteTM, Roche Diagnostics).
The beads were then washed extensively with ELB lysis buffer supplemented with 0.1mM PMSF, 1mM DTT, and a mixture of protease and phosphatase inhibitors, and then boiled for 5 minutes in 20 µL of Laemmli buffer.
The cells were lysed in TNE buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% NP-40 and 1 mM Na3VO4 containing a mixture of protease inhibitors).
The final pellet was diluted in Tris buffer and supplemented with a mixture of protease inhibitors (0.2 mM phenylmethylsulphonyl fluoride, 2 µg/mL leupeptin, and 0.5 µg/mL aprotinin) before it was aliquoted and frozen at −80°C.
Cells were lysed in ice-cold Mg2+ lysis/wash buffer (Upstate Biotechnology) containing mixture of protease and phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, complete protease inhibitor cocktail).
GST fusion proteins immobilized on glutathione Sepharose beads were incubated with rat brain synaptosomes (P2 fraction) in HKA buffer with 2% CHAPS and a mixture of protease inhibitors (Boehringer Mannheim) at 4°C for 12 hr.
They were gently detached with a rubber policeman and resuspended in 800 µl of homogenization buffer (10 mm triethanolamine, 10 mm acetic acid, 250 mm sucrose, 20 mm N-ethylmaleimide, 1 mm EDTA, and a mixture of protease inhibitors, pH 7.4).
rEA-containing fractions were treated with a mixture of protease-free RNase A (Boehringer, Columbus, OH) and DNase I (Roche, Indianapolis, IN), 4 hours at +4°C, 0.2 µg/ml, and 2 U/ml as final concentrations, respectively.
