Induction with LPS and TNF was performed on 1��106 ml astrocytes cultured research only for 24 hours with or with out 5 ug ml LPS or 50 ng ml TNF, with the superna tants collected for an MIP 2�� ELISA assay and the remaining cells washed three times with PBS and col lected for RNA or protein extraction. Astrocytes were transfected with the plasmid pAAV IRES hrGFP or the plasmid pAAV MIP 2�� hrGFP alone or combined with either the pBS U6 vector or the plasmid MIP 2�� siRNA using Polyfect according to the manufacturers instructions. Media were collected three days after transfection and the remaining cells were washed three times with PBS for RNA or protein extraction. The plasmid pAAV IRES hrGFP containing the hrGFP gene was used as a control for transient transfection.

The expression of hrGFP by astrocytes was analyzed by fluorescence microscopy using a ��20 objective, and the data were acquired with a Sony Inhibitors,Modulators,Libraries digital charge coupled device cam era and processed by Adobe PHOTOSHOP software. Inhibitors,Modulators,Libraries For neuron astrocyte co culture experiments, neurons were plated as normal in 24 well trays and maintained in culture for 11 days before addition of astrocytes. Transfected or untransfected astrocytes were plated in tissue culture inserts at 104 cells insert with 3. 0 um pores. Before assaying neuron survival, the inserts were removed and 3 2,5 diphenyl tetrazolium bromide assays or lactate de hydrogenase release tests were performed on the neurons. Immunostaining against the cytoskeleton con stituent, MAP 2, confirmed their Inhibitors,Modulators,Libraries neuron identity.

MIP 2�� ELISA Secreted, soluble MIP 2�� in culture supernatants was deter mined using a sandwich ELISA with Inhibitors,Modulators,Libraries a monoclonal Inhibitors,Modulators,Libraries anti mouse MIP 2�� antibody. The captured antibody was used at 1 ug ml and the biotinylated detection antibody was used at 400 ng ml. Detergent free isolation of lipid rafts Detergent resistant membranes from astrocytes were isolated on the basis of their insolubility in Triton X 100 at 4 C and their ability to float in density gradients as described previously. Astrocytes were homoge nized in a homogenizer in 1. 5 ml of PTN 50 buffer containing 10 mM dithiotheritol, 1 mM phenylmethylsulfonyl fluoride, 5 ug ml leupeptin, and 1 ug ml pepstatin A, and centrifuged at 12,000 rpm for 3 minutes. The supernatant was placed at the bottom of an ultracentrifugation tube and mixed with an equal vol ume of 80% sucrose.

The samples were overlaid with 30% and 5% sucrose, respectively. The gradient was centrifuged at 55,000 rpm for 2 hours at 4 C with a TLS 55 Beckmann swing rotor. Ten fractions were collected from the top of the gradient and protein levels quantified. An equal volume of each fraction www.selleckchem.com/products/ldk378.html was diluted in a loading buffer and used for im munoblotting. Fractions corresponding to raft microdo mains and fractions corresponding to detergent soluble material were pooled before loading on the same acrylamide gel.