Alternatively, RNA levels were analyzed using Illumina arrays. Total RNA (100?ng) was in vitro transcribed, amplified and biotin labeled using T7 oligo(dT) (Ambion TotalPrep RNA Amplification Kit) and hybridized to Illumina HT-12 arrays. The arrays were washed and stained with streptavidin-Cy3 and imaged on the Illumina iScan scanner. Data sets of triplicates were analyzed DEF6
using the Bioconductor package LIMMA with the Benjamini-Hochberg (FDR) test for multiple correction. Genes with an FDR-adjusted p-value of <0.05 were considered as differentially expressed. Datasets were also analyzed using the ArrayStar software package from DNAStar using the Students t-test and FDR correction with concordant results. Real-time PCR was performed with customized Taqman Array Plates (ABI) containing gene specific primers to assess induction of selected <a href="http://www.selleckchem.com/products/Cilomilast
(SB-207499).html">Cilomilast clinical trial genes. cDNA was prepared by reverse transcription of RNA samples (2??g) using Omniscript Reverse Transcriptase according to the manufacturer's instructions (Qiagen). The cDNA was adjusted to 100?ng/��L and 100?ng incubated with Taqman Gene Expression Master Mix containing AmpliTAq Gold Ultrapure DNA polymerase in a final volume of 20?��L per reaction. Plates were then analyzed on a RealPlex QPCR machine (Eppendorf). Relative increases in RNA were then analyzed using the Delta Delta Ct method  using GAPDH as the reference calibration gene. The region flanking the core bZip domain, termed the ATB domain (see text) was deleted by first amplifying a fragment by PCR from pDJB125 (containing CREB-H��TMC). Primers were designed to extend a fragment from the NheI site at the N-terminus of CREB-H��TMC and terminate at residue E229 followed by nucleotides corresponding to a BspEI site. Once amplified and digested with NheI and BspEI, the fragment was inserted back into pDJB125 similarly digested with NheI and BspEI, where the natural BspEI site encompasses E257. The result is deletion of residues 230�C257 within CREB-H��TMC, resulting in the plasmid pCB10 encoding CREB-H��TMC.��ATB. A version of this expressing CREB-H��TMC.��ATB under the control of the CMV promoter was also MK-4827
constructed by digesting pCB10 with BamHI and XbaI, and inserting the appropriate fragment into similarly digested pDJB150 [20, 22] to yield the vector pML28. COS cells were plated in 24-well dishes and transfected with the reporter plasmid (5XATF6-GL3) containing five repeats of the CRE-like UPR/ATF6 element . Cell lysates were prepared 24 or 48?h after transfection by addition of ��Glo Lysis Buffer�� according to the manufacturer's instructions (Promega). Luciferase activity was determined using the ��Bright-Glo�� luciferase assay system as described by the manufacturer. For comparison with the active form of CREB-H, we used a vector expressing the active form of ATF6 p3XFLAG-CMV-ATF6.