Aligned to the mouse genome (mm9 version) using ELAND. The sequences

Aligned to the mouse genome (mm9 version) using ELAND. The sequences were sent to our lab in the ELAND format. For the Bcl-3 ChIP we pooled two separate ChIP-seq experiments by combining.bam forms of the alignments. There were 40 million sequence reads in the control samples, of which 20.4 million were unique, and there were 52 million sequence reads in the unloaded samples of which 25.9 million were unique. The aligned sequences converted to.sam format were uploaded to the peak finder program in ChIPseeqer [15]. These alignment files were used for all subsequent analyses.reverse transcriptase (Applied Biosystems, Foster City, CA). mRNA expression was assessed 12926553 using TaqMan Gene Expression Assays and master mix (Applied Biosystems, Foster City, CA) detected by an ABI 7300 Real-Time PCR system as described previously [10]. Gene expression values were quantified by comparing CT values of the unknown sample to the gene-specific standard curve and normalized to the expression of beta-actin.Microarray Processing and AnalysisWhole-genome gene expression profiling experiments were carried out by the Boston University Microarray Core Facility. Each group (control and unloaded) included 4 independent total RNA samples with a minimal RIN number 8.0 verified by Bioanalyzer 2100 (Agilent Technology, Palo Alto, CA). Each total RNA sample was amplified, labeled, and hybridized on a mouse Affymetrix Gene 1.0 ST array (Santa Clara, CA) per manufacture instructions to measure expression of 28,853 well-annotated genes. A total of 8 array images were acquired by GeneChip Scanner 3000 7G and quality assessed by Affymetrix Expression Console (Santa Clara, CA). Gene expression signals were generated by robust multi-array analysis (RMA) [16] using Brainarray MoGene 1.0ST custom CDF files [17]. Differential gene expression was computed using the Comparative Marker Selection module in Genepattern database (Broad Institute, Cambridge, MA) which compares mean differences between control and unloaded groups by two-way parametric t-test. P-value #0.05 and q-value #0.05 were used to identify genes that were significantly differentially expressed with hind limb unloading. The microarray dataTotal RNA Isolation and RT-qPCRGastrocnemius and plantaris muscles harvested from anesthetized wild type mice from control and HU groups (n = 6 per group) were snap frozen in liquid nitrogen and stored at 280uC before use. Total RNA was isolated using the Qiagen miRNeasy Mini kit (Triptorelin site Valencia, CA) according to manufacturer’s instructions. Extracted total RNA was treated with RNase-Free DNase I (Qiagen, Valencia, CA), quantitated by UV spectrophotometry, and quality checked by a 1 denaturing agarose gel as previously described [10]. Five micrograms of total RNA was converted to cDNA in an 100 ml PCR reaction using random primers and MultiscribeA Bcl-3 Network Controls Muscle AtrophyTable 1. The genes from iPAGE ontology analysis.GO category Protein catabolism (11 GO terms)Gene Name Adam17 Arih2 Ate1 Cul2 Fbxo6 Hspa5 Itch Ppt1 Psen1 Rlim Sod1 Trim63 Ubr1 UspFunction Activates some membrane receptors E3 ligase Arginyl transferase Component of ECS ubiquitination E3 ligase Hsp 70 family purchase Indolactam V member E3 ligase Lysosomal degradation Intramembrane protein cleavage Ring finger protein Reactive radical destroyer Muscle E3 ligase (MuRF1) n-recognin for N rule proteolysis Ubiquitin thioesterase Wnt antagonist Sphingolipid recognition in lysosomes Wnt signaling glucose metabolism Essential for myogenin.Aligned to the mouse genome (mm9 version) using ELAND. The sequences were sent to our lab in the ELAND format. For the Bcl-3 ChIP we pooled two separate ChIP-seq experiments by combining.bam forms of the alignments. There were 40 million sequence reads in the control samples, of which 20.4 million were unique, and there were 52 million sequence reads in the unloaded samples of which 25.9 million were unique. The aligned sequences converted to.sam format were uploaded to the peak finder program in ChIPseeqer [15]. These alignment files were used for all subsequent analyses.reverse transcriptase (Applied Biosystems, Foster City, CA). mRNA expression was assessed 12926553 using TaqMan Gene Expression Assays and master mix (Applied Biosystems, Foster City, CA) detected by an ABI 7300 Real-Time PCR system as described previously [10]. Gene expression values were quantified by comparing CT values of the unknown sample to the gene-specific standard curve and normalized to the expression of beta-actin.Microarray Processing and AnalysisWhole-genome gene expression profiling experiments were carried out by the Boston University Microarray Core Facility. Each group (control and unloaded) included 4 independent total RNA samples with a minimal RIN number 8.0 verified by Bioanalyzer 2100 (Agilent Technology, Palo Alto, CA). Each total RNA sample was amplified, labeled, and hybridized on a mouse Affymetrix Gene 1.0 ST array (Santa Clara, CA) per manufacture instructions to measure expression of 28,853 well-annotated genes. A total of 8 array images were acquired by GeneChip Scanner 3000 7G and quality assessed by Affymetrix Expression Console (Santa Clara, CA). Gene expression signals were generated by robust multi-array analysis (RMA) [16] using Brainarray MoGene 1.0ST custom CDF files [17]. Differential gene expression was computed using the Comparative Marker Selection module in Genepattern database (Broad Institute, Cambridge, MA) which compares mean differences between control and unloaded groups by two-way parametric t-test. P-value #0.05 and q-value #0.05 were used to identify genes that were significantly differentially expressed with hind limb unloading. The microarray dataTotal RNA Isolation and RT-qPCRGastrocnemius and plantaris muscles harvested from anesthetized wild type mice from control and HU groups (n = 6 per group) were snap frozen in liquid nitrogen and stored at 280uC before use. Total RNA was isolated using the Qiagen miRNeasy Mini kit (Valencia, CA) according to manufacturer’s instructions. Extracted total RNA was treated with RNase-Free DNase I (Qiagen, Valencia, CA), quantitated by UV spectrophotometry, and quality checked by a 1 denaturing agarose gel as previously described [10]. Five micrograms of total RNA was converted to cDNA in an 100 ml PCR reaction using random primers and MultiscribeA Bcl-3 Network Controls Muscle AtrophyTable 1. The genes from iPAGE ontology analysis.GO category Protein catabolism (11 GO terms)Gene Name Adam17 Arih2 Ate1 Cul2 Fbxo6 Hspa5 Itch Ppt1 Psen1 Rlim Sod1 Trim63 Ubr1 UspFunction Activates some membrane receptors E3 ligase Arginyl transferase Component of ECS ubiquitination E3 ligase Hsp 70 family member E3 ligase Lysosomal degradation Intramembrane protein cleavage Ring finger protein Reactive radical destroyer Muscle E3 ligase (MuRF1) n-recognin for N rule proteolysis Ubiquitin thioesterase Wnt antagonist Sphingolipid recognition in lysosomes Wnt signaling glucose metabolism Essential for myogenin.