Additional controls on each array in cluded 6 positive controls consisting of Cy 3 labeled anti bodies, 6 negative controls containing bovine serum albumin, and empty spots containing no antibody, with the 6 replicates scattered throughout selleck screening library the array. The antibody microarray was performed according to the manufacturers protocol and slides were scanned using Full Moon Biosystems Scanning Array Service. Array data analysis Each spot throughout the array was scanned to provide its signal intensity values, including positive and negative controls. For background correc tion, the median value of the negative control signal was subtracted from the values of each antibody and phospho Inhibitors,Modulators,Libraries antibody. The background corrected signal was log2 trans formed and normalized by the mean value of beta actin signal.

For each phosphorylated antibody, Inhibitors,Modulators,Libraries the background corrected signal was normalized by the mean value of the corresponding total antibody. The fold changes between treatment groups were derived from ratios of geometric mean signal intensities. Treatment groups included con trol non infected monocytes co cultured with HBMEC, HIV infected monocytes co cultured with HBMEC, and HIV infected monocytes treated with CCR5 blockers and co cultured with HBMEC. ANOVA with heterogeneous variance was used for statistical analyses of protein expression between groups, and the Tukey Kramer method used for multiple compar isons. The Benjamini Hochberg method was then used to control the false discovery rate. Proteins with BH adjusted p value less than 0. 05 were considered to be dif ferentially expressed.

Ingenuity pathway analysis Differentially expressed and phosphorylated proteins identi fied in HIV 1 infected monocytes co cultured with HBMEC, compared Inhibitors,Modulators,Libraries to non infected monocytes co cultured with HBMEC, or infected monocytes treated with CCR5 blockers and co cultured with HBMEC, were analyzed using the In genuity Pathways Analysis 3. 0. The networks obtained through IPA software describe func tional relationships between proteins products based on known interactions, biological functions and canonical path ways. Using a false discovery rate of 0. 05, only phospho proteins upregulated or downregulated by at least 1. 5 fold were considered. Inhibitors,Modulators,Libraries and for non phosphorylated proteins, only those upregulated or downregulated by at Inhibitors,Modulators,Libraries least 2 fold were considered. Monocyte adhesion www.selleckchem.com/products/Nilotinib.html to an in vitro BBB model For adhesion assays, HBMEC were plated on 96 well collagen coated black bottom plates and cultured to con fluence. Infected monocytes treated or non treated with CCR5 blockers or CCR5 antibodies were labeled with 5 carboxyfluorescein diacetate, acetoxymethyl ester, 10 uM1106 for 1 hour, and co cultured with HBMEC for 15 min.