Paclitaxel 2 and Neuro. 2A cells were harvested and in vivo, mouse hippocampal tissue was unthawed, and lysed in ice cold lysis buffer containing, 100 mM HEPES, 150 mM NaCl, 1% Nonidet P 40, 2 mM EGTA, 2 mM Sodium Ortho vanadate, Protease Inhibitor cocktail, and 1 mM PMSF and centrifuged at 11000 �� g for 10 Inhibitors,Modulators,Libraries min at 4 C to remove all cellular debris. Protein concentration was determined using the BCA Protein Assay according to the manufacturers protocol. Lysate concentration was then normalized and denatured in SDS PAGE buffer at 95 C and stored at 20 C until use. All lysates were electro phoresed and separated on a 7. 5% SDS PAGE gel, and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non fat milk and incubated with anti phosphory lated STAT3 antibody overnight at 4 C.
After incubation with an HRP conjugated secondary antibody, the protein bands were detected with a chemiluminescenct substrate and Bio Max film. For detection of total STAT3 protein, the membranes Inhibitors,Modulators,Libraries were stripped with stripping buf fer, 0. 70% b ME followed by overnight incubation with anti STAT3 anti body at 4 C. Immunoblot results were quantified using ImageJ 1. 41 software. Cytokine detection in cell supernatant, hippocampus, and plasma Hippocampal tissue was lysed in ice cold lysis buffer and protein concentrations were determined using the BCA protein assay according to manufacturers proto col. For hippocampal tissue, the antibodies and stan dards for the IL 6 ELISA were used according to the description by the manufacturer.
Cell supernatants and plasma samples were assayed for IL 1b, TNF a, IL 6, and the anti inflamma tory cytokine IL 10, using multiplexed bead based immunoassay kits combined with a Cytokine Reagent kit as described by the manufacturer. Cytokine mRNA measurement Inhibitors,Modulators,Libraries by quantitative real time PCR Total RNA from hippocampus was Inhibitors,Modulators,Libraries isolated using the Tri Reagent protocol A Quanti Tect Reverse Transcription Kit was used for cDNA synthesis with integrated removal of genomic DNA contamination according to the manufac turers protocol. Quantitative real time PCR was per formed using the Applied Biosystems Assay on Demand Gene Expression protocol as pre viously described. In brief, cDNA was amplified by PCR where a target cDNA and a reference cDNA were amplified simultaneously using an oligonucleotide Inhibitors,Modulators,Libraries probe with a 5 fluorescent reporter dye and a 3 quencher dye.
PCR reactions were performed in triplicate under the following conditions, 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. Fluorescence was determined on an ABI PRISM 7900HT sequence detection system. Data were analyzed sellectchem using the comparative threshold cycle method, and results are expressed as fold difference. Statistical analysis All data were analyzed using Statview and Statistical Analysis System software.