The probe was then pipetted onto the printed surface of the slide. Tofacitinib Citrate A coverslip was carefully placed on top of the array to avoid bubble formation during hybridization. The chamber was placed in a 42 C water bath for 16 hours. Post hybridization washing The array was washed in 2�� SSC, 0. 1% SDS at 42 C for 5 minutes, and then in a second buffer containing 0. 1�� SSC, 0. 1% SDS at room temperature for 5 minutes, and the process was repeated once. The array was then washed 4 times in 0. 1�� SSC buffer at room temperature for 1 minute. The array was then dried by centrifugation, and the signal emitted from each spot was analyzed with digital imaging software. Western blot analysis Total proteins were extracted from test THP 1 cells with ice cold lysis buffer, 20 mM EGTA, 1 mM dithiothreitol, and protease inhibitor cocktail and centrifuged at 12,000 �� g for 20 min.

Protein sam ples were subjected to western blotting as described pre viously. 9 Briefly, test proteins were assayed after overnight incubation at 4 C with 1,1000 dilution of poly clonal p44 p42 MAPK or phosphor specific ERK1 2 antibodies. Equal protein load ing was assessed using mouse a actin. The proteins were visualized with an enhanced chemiluminescence detection kit. Data and signaling pathways analysis The focused array system that we used in this study was adapted from the system reported by Iyer et al. and Wang et al. We employed Cy3 and Cy5 fluorescent dyes to label the RNA samples obtained from the control and treatment groups, respectively.

The Cy3 and Cy5 labeled RNA samples were then mixed and subjected to hybrdization with oligo nucleotide probes on chips. Five different house keeping genes, alpha Tubulin, beta 2 microglobulin, beta actin, GAPDH, Transferrin R, have been built into the design of our array genes. These 5 housekeeping genes were hence employed as the internal controls of our gene chip assay. Within each array chip, four replicates for each gene were used. The scanning output generated from the focused arrays was fed into GenePix to extract numerical expression readings from each spot. The relative expression level of each gene was represented by the median of ratio averaged from the four replicates of a gene on the same array. As we pre viously described, our microarray data were ana lyzed using the Spotfire software, which includes established algorithms that determine whether a gene is present or absent and whether Brefeldin_A the expression level of a gene in specific experimental test samples is sig nificantly increased or decreased relative to a control sample, and for clustering distinct groups of gene expression profiles. The signals obtained from different chips were normalized by the relative expression level to the b actin gene.