drugdisc © 00 Macmillan Publishers Limited. All rights reserved Topical formulation. NF B Confers IL6 Independence Several such proteins, such as the SV40 large and small T anti infections were carried out in the presence of 8 g/ml poly gens and the human Letrozole papillomavirus E6 and E7 proteins, have been successfully used as molecular tools to discern the role of cellular signaling pathways in various biological processes 8. The human herpesvirus 8 HHV8, also known as Kaposi’s sar comaassociated herpesvirusencoded K3 protein contains two tandem death effector domains that are also present in the brene Sigma. Postinfection, cells were cultured in normal growth media containing the appropriate drugs to select posi tive clones or sorted based on GFP fluorescence. Cell Viability and Cell Cycle Assays

Cells from exponen tially growing cultures were washed three times with human IL6 free medium and plated in an untreated flatbottom 6well prodomain of caspase 8/FLICE. Proteins with two death effec plate at a density of 5 0 3 cells/well in the presence or Letrozole CGS 20267 absence tor domains are also found in several other viruses and include MC5L and MC60L from the Molluscum contagiosum virus and E8 from equine herpesvirus  EHV  . These pro teins were originally believed to protect virally infected cells from death receptorinduced apoptosis by blocking the recruit ment and/or activation of caspase 8/FLICE and as such were collectively referred to as viral FLICE inhibitory proteins or vFLIPs  . However, subsequent work by our laboratory and others showed that K3 does not act as a vFLIP but instead directly interacts with the NEMO/IKK subunit of the IKK complex to selectively activate the NF B pathway 4. In this study, we have taken advantage of this unique ability of K3 to selectively activate the NF B pathway and used it as a molecular tool to study the role of the NF B pathway in IL6 independent growth of murine plasmacytoma cells. MATERIALS AND METHODS Cell lines and Reagents

T65 and B cells were grown in RPMI medium supplemented with0% v/v FCS,00 units/ml penicillin,00 g/ml streptomycin, m M sodium pyruvate,  m M glutamine all from Invitrogen, and0 ng/ml and 5 ng/ml of recombinant human IL6 PeproTech, Inc., respectively. HEK3FT cells Invitrogen were grown in Dulbecco’s mod ified Eagle’s medium supplemented with0% v/v fetal bovine of hIL6. Cell viability was measured after 48 h using the MTS Letrozole Aromatase inhibitor reagent 3 4,5dimethylthiazolyl53carboxymethoxy phenyl4sulfophenylHtetrazolium, inner salt follow ing manufacturer’s instructions Promega, Madison, WI. Absorbance of viable cells was measured at 40 nm with 600 nm as a reference wavelength. Percent cell survival was calculated based on the reading of cells grown in the presence of hIL6 as00%. DNA content analysis was performed as described pre viously 5. ELISA for Murine IL6  Cells were harvested by centrifuga tion, washed three times in medium lacking growth factors, and then set up at0 5 cells/ml in IL6free medium for 7 h.

Cells were centrifuged, and the supernatant was filtered and assayed for mIL6 using an IL6 ELISA kit eBioscience following the recommendations of the manufacturer. Assays for Nuclear NF B DNAbinding Activity  Nuclear proteins were extracted and used for measuring the status of NF B DNA binding by EMSA or an ELISAbased transfector kit Clontech, as described previously , 7. Western Blot Analysis  Cells were lysed in a lysis buffer con taining 0 m M sodium phosphate pH 7.4,50 m M NaCl, 0.% Triton X00, 0. M ununseptium PMSF, and0% glycerol supplement with a protease inhibitor mixture tablet Roche. Western blot analy sis was performed essentially as described previously 5. Pri serum,00 units/ml penicillin,00 g/ml streptomycin, and mary antibodies used in these experiments were FLAGHRP 50 g/ml Geneticin sulfate in a humidified atmosphere con taining 5% CO  at 37 °C. Dexame