Protein concentrations in the superna tants were determined in duplicate using the Bradford protein assay. Mice were housed and treated in accord ance with National Institutes of Health and the University of Alabama at Birmingham Institutional CP127374 Animal Care and Use Committee guidelines. Cell culture, immunoblotting, and cell viability assay For murine primary neurons, the hippocampus from 1 day old C57Bl 6 mice was isolated and incubated in 0. 1% trypsin and the cells mechanically dissoci ated using a fire polished pipette. Cells were plated in DMEM F12 medium supplemented with 10% FBS, 0. 3% glucose, 2 mM glutamine, 10 U mL penicillin and 10g mL streptomycin in poly D lysine coated six well plates. Twelve hours after plating, the medium was replaced with Neurobasal medium supplemented with B27 and 0.

Inhibitors,Modulators,Libraries 5 mM glutamine to promote neuronal survival and to inhibit the growth of non neuronal cells. Neurons were used for experiments after seven days in culture. Primary glia were prepared from the cerebral cortex of 1 day old C57Bl 6 mice as described, cultured in DMEM F12 medium supplemented with 10% FBS, 0. 3% glucose, 2 mM L glutamine, 10 U mL penicillin and 10g mL strep tomycin. For separation Inhibitors,Modulators,Libraries of astrocytes and microglia, after 10 days of culture the cells were shaken, resulting in 99% pure astrocytes as determined by immunostaining with the astrocyte marker glial fibrillary acidic protein. After the first hour of shaking, the medium containing microglia cells was collected and microglia were cultured in the same medium as astrocytes.

For expression experiments, cells were rinsed twice with DMEM F12 medium without supplements, infected with 50 moi of the Inhibitors,Modulators,Libraries designated adenovirus Inhibitors,Modulators,Libraries for 30 min, supple mented medium was added for incubation for 36 48 h, and infected cultures were examined for adequate infec tion efficiency as assessed by GFP fluorescence after infection with GFP adenovirus. For knockdown experiments, cells were transfected using liposome medi ated transfection reagent LipofectAMINE RNAiMAX with 50 nM siRNA according to the manufacturers instructions with STAT3 prevalidated siRNA, silencer Inhibitors,Modulators,Libraries negative control, or GSK3 and GSK3 siRNA. For experi mental treatments, cells were pretreated with the indi cated inhibitors for 30 min followed by treatment with 100 ng mL LPS, 1 ng mL IFN, or both, for the indicated times.

Following treatments, cells were washed twice with phosphate buffered saline and were lysed with lysis buffer. The lysates were centrifuged at 14 000 rpm for 10 min. Protein concentrations were determined in duplicate using the Bradford protein assay. Immunob selleckbio lotting was carried out as described before using anti bodies to phospho Tyr705 STAT3, total STAT3, GFAP, GSK3, and actin. Immunoblots were developed using horseradish peroxidase conjugated goat anti mouse, or goat anti rabbit IgG, followed by detection with enhanced chemiluminescence, and the protein bands were quanti tated with a densitometer.