Protein concentration was determined using the Pierce BCA Protein assay kit, as per the manufacturers instruc tions. For western blotting, 20g cytoplasmic protein was loaded into 10% polyacrylamide gels containing SDS and separated by electrophoresis. Proteins were transferred onto Protran nitrocellulose membranes by electroblotting and were stained with Pon ceau S to qualitatively determine equal loading of samples and efficient transfer of proteins. Membranes were blocked in 5% nonfat milk in 0. 05% Tris buffered saline containing 0. 05% Tween 20 for 1 hour followed by incubation with primary antibodies in block ing buffer overnight. Membranes were washed in TBST and incubated in 5% milk TBST with appropriate secondary anti body for 45 minutes to 1. 5 hours.
Membranes were then washed with TBST and rinsed in Tris buffered saline prior to incubation in Supersignal West Pico Chemiluminescent Sub strate and exposed to Amer sham Hyperfilm ECL. Membranes were stripped using Panobinostat ic50 1 M glycine, pH 2. 5, and washed using TBST prior to reprobing. RNA isolation Total RNA was isolated from cultures by Trizol fol lowed by RNeasy clean up as per the manufacturers directions. Total RNA was quantified. High quality RNA for use in the microarray analysis was confirmed by analysis in the Agi lent 2100 Bioanalyzer. Microarray analysis Total mRNA from two biological replicates of cells treated with DMSO, U0126, TNF or U0126 and TNF, were amplified once and hybridized to RAT2302. 0 gene chips. Amplification, labelling, hybridization and detection were performed at the London Regional Genomics Centre according to the manufacturers instructions.
Microarray data and gene ontology analysis The raw expression values were imported into Genespring GX 7. 3. Raw expression values 0. 01 were set to 0. 01 and the normalization natural product libraries per chip was set to the 50th percentile. Relative gene expression of the 31,099 probe sets on the chip was determined by normalizing the raw expression values for each probe set to the DMSO control from each independent experiment. To identify genes that were TNF regulated, probe sets that were altered 1. 45 in DMSOTNF treated cultures compared with DMSO treated cultures were deter mined for each independent experiment. Probe sets identified as being TNF regulated in both independent experiments were selected for further analysis. Genes whose transcript lev els changed 1.
45 fold were selected for study, as our micro array analysis revealed that aggrecan mRNAa transcript previously shown to be TNF sensitivewas reduced approximately 1. 45 fold and thus served as a positive control establishing the validity of our microarray data. To identify probe sets whose changes were altered by TNF in a MEK12 dependent fashion, we normalized the fold change in gene expression of U0126TNF treated cultures to that of cultures treated with U0126 alone from both inde pendent experiments.