The magnetic separator to select glomeruli containing Dynabeads. Purified glomeruli were

The magnetic separator to select glomeruli containing Dynabeads. Purified glomeruli were further digested with 5 mg/ml of type V Collagenase (Sigma) at 37uC for 30 minutes with agitation, magnetically separated, and then Digested glomeruli were centrifuged, resuspended and incubated with PECAM-1 coated Dynabeads. After washing all materials with HBSS for 5 times, recovered endothelial cells from all organs were lysed with buffer RLT plus for RNA preparation, with RNeasy Micro Plus kit (Qiagen).only the genes with maximum expression values across all microarrays greater than 200 were analyzed here. The genes with p,0.01 and fold change .4 were considered as specifically expressed. The combination of p value and fold change threshold serves to eliminate most false positives, as suggested from a large microarray study led by FDA [208]. Fisher’s exact test was used to identify the potential enriched pathways from these brain endothelial specific genes.Protein-protein Interaction (PPI) NetworksPPI datasets for human and mouse were downloaded from BIOGRID database at version 3.1.71. Since there were only 2314 58-49-1 web proteins and 4118 interactions in the mouse PPI dataset, we transformed human PPI information into mouse’s based on the homolog genes between human and mouse according to NCBI HomolGene database. The human PPI dataset contained 10121 proteins and 52693 interactions. After combining the native mouse dataset and the transformed mouse dataset and deleting repeated records and self-self interaction records, a mouse PPI network with 9189 proteins and 36073 interactions was built. Since not all proteins in the networks are expressed in the endothelial cells under this study, we further shrink the network to EC-specific PPI (EC-PPI) network by deleting the proteins that are not expressed and their corresponding interactions. The EC-PPI contains 4243 proteins and 10825 interactions. The properties of network were calculated with IGRAPH package in R. The PPI networks were visualized by Cytoscape software with force-directed layout.Real Time PCRRelative expressions of selected markers for different types of cells were tested with RT-PCR, with pre-designed primers and Syber Green system from Bioscience. First strand cDNA was synthesized with QuantiTect reverse transcription system (Qiagen). Date normalization was performed by quantification of the endogenous 18S rRNA, and fold change was measured with 22DDCt method. The markers for endothelial cells included C.I. 19140 VE-cadherin, PECAM-1 and eNOS. To ensure that our brain vasculome was not contaminated by parenchymal non-endothelial cells, we also checked markers for astrocytes (Aquaporin-4, GFAP), markers for neurons (MAP-2, Neurogranin), and markers for pericytes and smooth muscle cells (smooth muscle alpha-Actin Acta2), calponin 1 CNN1, 1527786 desmin, myosin heavy polypeptide 11 Myh11, transgelin Tagln). For heart and glomerular preparations, we checked markers for myocytes (Myh6, NKX 2?), markers for glomerular podocytes (Nphs-1, Nphs-2) and markers for kidney tubules (Cadherin-16, Claudin16, Lrp2).GWAS and Plasma Protein DatabasesGenome-wide- association-studies of disease select the risk genes for the disease. GWAS-identified disease genes for stroke, Alzheimer’s disease and Parkinson’s disease were collected (dbGAP: http://www.ncbi.nlm.nih.gov/projects/gapplusprev/ sgap_plus.htm) to analyze the expression of such disease-related genes in endothelial cells. The expression of human plasma proteins were also.The magnetic separator to select glomeruli containing Dynabeads. Purified glomeruli were further digested with 5 mg/ml of type V Collagenase (Sigma) at 37uC for 30 minutes with agitation, magnetically separated, and then Digested glomeruli were centrifuged, resuspended and incubated with PECAM-1 coated Dynabeads. After washing all materials with HBSS for 5 times, recovered endothelial cells from all organs were lysed with buffer RLT plus for RNA preparation, with RNeasy Micro Plus kit (Qiagen).only the genes with maximum expression values across all microarrays greater than 200 were analyzed here. The genes with p,0.01 and fold change .4 were considered as specifically expressed. The combination of p value and fold change threshold serves to eliminate most false positives, as suggested from a large microarray study led by FDA [208]. Fisher’s exact test was used to identify the potential enriched pathways from these brain endothelial specific genes.Protein-protein Interaction (PPI) NetworksPPI datasets for human and mouse were downloaded from BIOGRID database at version 3.1.71. Since there were only 2314 proteins and 4118 interactions in the mouse PPI dataset, we transformed human PPI information into mouse’s based on the homolog genes between human and mouse according to NCBI HomolGene database. The human PPI dataset contained 10121 proteins and 52693 interactions. After combining the native mouse dataset and the transformed mouse dataset and deleting repeated records and self-self interaction records, a mouse PPI network with 9189 proteins and 36073 interactions was built. Since not all proteins in the networks are expressed in the endothelial cells under this study, we further shrink the network to EC-specific PPI (EC-PPI) network by deleting the proteins that are not expressed and their corresponding interactions. The EC-PPI contains 4243 proteins and 10825 interactions. The properties of network were calculated with IGRAPH package in R. The PPI networks were visualized by Cytoscape software with force-directed layout.Real Time PCRRelative expressions of selected markers for different types of cells were tested with RT-PCR, with pre-designed primers and Syber Green system from Bioscience. First strand cDNA was synthesized with QuantiTect reverse transcription system (Qiagen). Date normalization was performed by quantification of the endogenous 18S rRNA, and fold change was measured with 22DDCt method. The markers for endothelial cells included VE-cadherin, PECAM-1 and eNOS. To ensure that our brain vasculome was not contaminated by parenchymal non-endothelial cells, we also checked markers for astrocytes (Aquaporin-4, GFAP), markers for neurons (MAP-2, Neurogranin), and markers for pericytes and smooth muscle cells (smooth muscle alpha-Actin Acta2), calponin 1 CNN1, 1527786 desmin, myosin heavy polypeptide 11 Myh11, transgelin Tagln). For heart and glomerular preparations, we checked markers for myocytes (Myh6, NKX 2?), markers for glomerular podocytes (Nphs-1, Nphs-2) and markers for kidney tubules (Cadherin-16, Claudin16, Lrp2).GWAS and Plasma Protein DatabasesGenome-wide- association-studies of disease select the risk genes for the disease. GWAS-identified disease genes for stroke, Alzheimer’s disease and Parkinson’s disease were collected (dbGAP: http://www.ncbi.nlm.nih.gov/projects/gapplusprev/ sgap_plus.htm) to analyze the expression of such disease-related genes in endothelial cells. The expression of human plasma proteins were also.