natant was removed and again separated by cen trifugation at 100,000 g for 30 minutes at 4 C. The obtained pellets contained the total cytoplasmic membranes and were resuspended in 5% SDS with 0. 1 mmol l of PMSF and fi nally, after determination of protein density using the bicinchoninic STI 571 Inhibitors,Modulators,Libraries acid method, diluted in SDS PAGE buffer, 30% glycerol, 10% SDS, 0. 6 mol l DTT and 0. 012% of bromophenol blue and boiled at 95 C during 5 minutes for western blotting analysis. Preparation of hippocampal synaptosomes The preparation of hippocampal synaptosomes from rats was carried out essentially as described previously. After removal of the brain, the dissected hippocampi were homogenized in the same sucrose solution described above, and the homogenates were separated by centrifugation at 3,000 g for 10 minutes at 4 C.
The supernatant was removed Inhibitors,Modulators,Libraries and again separated by centrifugation at 14,000 g for 12 minutes at 4 C. The resulting pellet was resuspended in 1 ml of a 45% Percoll solution prepared in a Krebs HEPES Ringer solution at 4 C. This hom ogenate was then separated by centrifugation at 12,650 g for 2 minutes at 4 C in an Eppendorf microcentrifuge. The resulting white top layer was col lected, resuspended in 1 ml of KHR, and separated by cen trifugation at 12,650 g for 2 minutes at 4 C. The pellets were resuspended and diluted in the same solutions as total cytoplasmic membranes for western blot ting analysis. Preparation of sub synaptic membrane fractions To gauge the sub synaptic localization of IL 1B receptors, we used purification of sub synaptic membrane fractions, following the method initially published by Phillis et al.
and adapted by our group. This method can separate, with over 90% efficiency, membrane proteins from the ac tive zone 25 the cytoskeletal post synaptic density, and the non active zone fraction. This sub synaptic fractionation begins Inhibitors,Modulators,Libraries with the purifica tion of synaptosomes. Inhibitors,Modulators,Libraries For this purpose, the hippocampi were homogenized in 2. 5 ml of isolation buffer, and 1 ug ml of a prote ase inhibitor cocktail, and 100 ul of this mi ture was stored at ?80 C for later analysis. The homogenate was transferred to 50 ml centrifuge tubes AV-951 at 4 C, and 12 ml of a 2 mol l sucrose solution was added, together with 5 ml of 0. 1 mmol l CaCl2 to yield a final solution with 1. 25 mol l sucrose. This was then divided into two tubes and 2.
5 ml of 1 mol l sucrose solution was carefully layered over the solution in each tube. The tubes were equilibrated with isolation buffer and separated by centrifugation at 100,000 g for 3 hours at 4 C. The synaptosomes were captured at the interface between the 1. 25 mol l and 1 mol l sucrose solutions, and were then diluted 1 10 in isolation buffer. After centrifugation at 15,000 g for selleckchem Ruxolitinib 30 minutes at 4 C the pellet was resuspended in 1. 1 ml isolation buffer, and 100 ul of this mi ture was stored at ?80 C for later analysis. For preparation of the various sub synaptic fractions, the synaptosomes were diluted in 10 ml of a