Midazolam and propofol were obtained from Sigma Aldrich Chemical Co. Wedelo lactone, SP600125, PD98059 and Janus family of tyro sine kinase inhibitor I were obtained from Calbiochem selleck catalog Novabiochem Co. Phospho specific Inhibitors,Modulators,Libraries p38 mitogen activated protein kinase, p38 MAP kinase, phospho specific stress activated pro tein kinase c Jun N terminal kinase, SAPK JNK, phospho specific inhibitory kappa B, I B, phospho specific signal transducer and activator of tran scription 3 and STAT3 antibodies were purchased from Cell Signaling. Glyceral dehyde 3 phosphate dehydrogenase antibo dies were purchased from Santa Cruz Biotechnology, Inc. An enhanced chemiluminescence Western blotting detection system was obtained from GE Healthcare UK. Ltd. Other materials and chemicals were obtained from Inhibitors,Modulators,Libraries com mercial sources.

Wedelolactone, SP600125, PD98059 and JAK inhibitor I were dissolved in dimethyl sulfoxide. Inhibitors,Modulators,Libraries Propofol was dissolved in ethanol. The maximum con centration of dimethyl sulfoxide or ethanol was 0. 1%, which did not affect the assay for IL 6 or Western blot analysis. The viability of cells with 0. 1% dimethyl sulfox ide or ethanol treatment after 36 h was above 97% com pared to the cells without treatment by trypan blue staining. Cell culture Rat C6 glioma cells, obtained from the American Type Culture Collection, were seeded into 35 mm or 90 mm diameter dishes and maintained in Dulbeccos modified Eagles medium containing 10% fetal bovine serum at 37 C in a humidified atmosphere of 5% CO2 95% air. The medium was exchanged for serum free DMEM after 6 days. The cells were then used for experiments after 24 h.

The cells were pretreated with midazolam, propofol, wedelolactone, SP600125, PD98059 or JAK inhibitor I for 60 min before IL 1b sti mulation when indicated. Inhibitors,Modulators,Libraries Assay for IL 6 Cultured cells were stimulated with 10 ng ml IL 1b in serum free Inhibitors,Modulators,Libraries DMEM for 36 h. The conditioned medium was collected at the end of the incu bation, and IL 6 concentration was measured using an ELISA kit. The absorbance of each sample at 450 nm and 540 nm was measured with a Multiscan JX ELISA reader. Absorbance was corrected with reference to a standard curve. Western blot analysis Cultured cells were stimulated with 10 ng ml IL 1b in serum free DMEM for the indi cated periods. sellekchem The cells were washed twice with phos phate buffered saline and then lysed and sonicated in a lysis buffer containing 62. 5 mM Tris HCl, 2% sodium dodecyl sulfate, 50 mM dithiothreitol, and 10% glycerol. The sample was used for Western blot analysis. The samples were separated by SDS polyacryla mide gel electrophoresis using the method of Laemmli in 10% polyacrylamide gels.