Stimulated with LPS (one hundred ng/ml in development medium) for 4 h. The cultured media were then collected and spun down for five min at 2000 rpm, plus the concentrations of IL-1 , IL-6, and IFN within the medium were determined by ELISA using specific monoclonal antibodies and the procedures suggested by the suppliers (R D Systems, Minneapolis, MN and PBL Interferon Supply, Piscataway, NJ). The serum in the culture media did not interfere with all the assays. Whenever CB1 or CB2 receptor antagonists (SR141716 and SR144528, respectively) or abn-CBD were applied, they had been added 30 min before the beginning of the THC or CBD therapy. Western Blot Analysis–To examine the levels of IL-1 receptor-associated kinase 1 (IRAK-1) and of I B proteins and of the phosphorylated kind of the p65 NF- B subunit, BV-2 cells had been incubated with THC or CBD at 1, 5, or 10 M. Two h later the cells had been stimulated for 15 min with 100 ng/ml LPS. The cells were then rinsed twice with ice-cold PBS and lysed with RIPA buffer (140 mM NaCl, 20 mM Tris, pH 7.4, ten IL-2 Formulation glycerol, 1 Triton X-100, 0.5 sodium deoxycholate, 0.1 SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and leupeptin at 20 g/ml). Lysates have been centrifuged at four (ten min, 14,000 rpm) and pellets discarded, along with the supernatants have been aliquoted and stored at 20 for further evaluation. Aliquots of 25 g of proteins (as measured using the Bradford protein assay) from every single sample were separated by 10 SDSPAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5 nonfat milk in 10 mM Tris-HCl, pH 7.6, containing 150 mM NaCl and 0.5 Tween 20 (TBST). The blots have been incubated overnight at 4 with principal antibodies, like rabbit anti-IRAK-1, rabbit anti-I B (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-p65 (Ser-536) (Cell Signaling, Danvers, MA), or basic rabbit antip65 subunit of NF- B (Santa Cruz Biotechnology). Just after in depth wash with TBST, horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) was applied for 1 h at room temperature, along with the blots have been extensively washed and visualized applying an enhanced chemiluminescence detection kit (EZ-ECL Biological Industries). The blots had been scanned and quantified with NIH Image 1.63. The intensity of the staining of -actin (employing anti- -actin monoclonal antibody, Santa Cruz Biotechnology)JOURNAL OF BIOLOGICAL CHEMISTRYEXPERIMENTAL PROCEDURES Reagents–LPS (Escherichia coli serotype 055:B5) and Sigma Receptor Agonist Gene ID propidium iodide (PI) have been bought from Sigma. THC and CBD had been obtained from the National Institute on Drug Abuse (Baltimore, MD). SR141716, SR144528, and abn-CBD were obtained from Tocris (Ellisville, MO). Stocks of those supplies in ethanol or DMSO had been kept at 80 and diluted into medium just prior to experiments. Final concentration of ethanol or DMSO in culture medium was 0.1 . At this concentration, ethanol or DMSO did not show any considerable effect on the investigated parameters. Microglial Cell Culture–The BV-2 murine microglial cell line, originally generated by E. Blasi (University of Perugia, Perugia, Italy (see Ref. 11)), was kindly offered by Prof. E. J. Choi from the Korea University (Seoul, Korea). The BV-2 cells were cultured at 37 inside a humidified atmosphere of 95 air and 5 CO2 in higher glucose Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 5 heat-inactivated fetal bovine serum, streptomycin (one hundred g/ml), and penicillin (100 units/ml) (Biol.