MSB 1 cells were grown in Leibowitzs L 15 and McCoy 5A media supplemented with fetal bovine serum, penicillin at 37 C. Cells were cross linked with formaldehyde, which was added directly to the culture medium. The culture medium was removed and washed twice with ice cold phosphate buffer saline containing Regorafenib VEGFR inhibitor protease inhibitor cocktail. ChIP was done using the Chromatin Immunoprecipitation Assay kit exactly following manufacturers recommendations. Immunoprecipitation was performed with anti Meq polyclonal antibody, incubated overnight at 4 C. The DNA Meq antibody complexes Inhibitors,Modulators,Libraries were purified using Protein A agarose salmon sperm DNA beads. The purified complex sample was reverse cross linked separating the DNA from Meq and its interacting proteins.

Inhibitors,Modulators,Libraries Proteins that were co immunoprecipitated with Meq were analyzed and identi fied by 2D LC ESI MS MS as described above. Plasmid construction The CD30 promoters of six different chicken lines were amplified by PCR with Pfu poly merase and primers CD30 F and CD30 R. The amplified promoters were ligated into pCRW2. 1 TOPOW producing pCRW2. 1 CD30 plasmids. The cytomegalovirus promoter in the pd2EGFP N1 plasmid was removed by digestion with XhoI and VspI, linear DNA was blunt ended by T4 DNA polymerase and then self ligated pro ducing pd2EGFPCMV. CD30 promoters were released from the pCRW2. 1 CD30 plasmids by EcoRI digestion and ligated into EcoRI linearized pd2EGFPCMV resulting in production of the six new expression plas mids pd2EGFP CD30. The Meq promoter of the virulent MDV 1 strain RB 1B was amplified by PCR with primers MEQ F and MEQ R.

The promoter was first cloned into pCRW2. 1 TOPOW, then released by EcoRI digestion and re cloned into EcoRI linearized Inhibitors,Modulators,Libraries pd2EGFPCMV producing the re porter plasmid pd2EGFP Meq. The chicken cDNA en coding the NFB p100 was released from the cloning vector pBS KS with HindIII and XbaI and inserted into HindIII and XbaI linearized expression vector pBK CMV, resulting in pBK CMV p100. The cDNA encoding the chicken NFB p105 cloned in pGEM4 was released by diges tion with EcoRI and KpnI and inserted into EcoRI and KpnI linearized pBK CMV, producing pBK CMV p105. The ankyrin repeats were removed from the 5 end of the NFB p105 cDNA by digestion with SacI. The chicken NFB p65 cDNA cloned in pTZ18R was released by digestion with XhoI and MfeI and re cloned into Inhibitors,Modulators,Libraries XhoI and SmaI linearized pBK CMV producing pBK CMV p65.

Plasmids were purified using the Inhibitors,Modulators,Libraries affinity chromatography columns and proper structure of all the plasmids was verified by restriction enzymes digest and sequencing. Promoter assays The activity of CD30 and Meq promoters was analyzed in vitro by promoter reporter CHIR99021 GSK-3 inhibitor assays. First, the reporter gene d2EGFP was placed under the control of the CD30 and Meq promoters and the coding sequences of tran scription factors were cloned into the expression plasmid pBK CMV.