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Synthesis, secretion, and subcellular localization of serglycin proteoglycan in human endothelial cells by Barbara P. Schick, Joel F. Gradowski, and James D. San Antonio Blood Volume 97(2):449-458 January 15, 2001 ©2001 by American Society of Hematology Detection of serglycin mRNA in 3 types of endothelial cells.The cell cultures, mRNA extractions, and RT-PCR reactions were carried out as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Characterization of HUVEC-secreted and HUVEC-associated proteoglycans and their chondroitinase and heparitinase digestion profiles.Proteoglycans were purified as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Core proteins of endothelial cell–secreted proteoglycans.Digestions were performed as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Analysis of the secreted endothelial proteoglycans.(A) Fractionation of the secreted proteoglycans for analysis. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Recombinant serglycin and immunoblotting by chicken antiserglycin IgY.The recombinant serglycin was electrophoresed on 4% to 20% SDS-PAGE gradients and immunoblotted onto nitrocellulose. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Immunoblotting and autoradiographic detection of serglycin in HL-60 cells.HL-60 cells were incubated with 35S-sulfate, and proteoglycans were purified and subjected to chondroitinase digestion as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Immunoblotting of serglycin core protein from secreted and intracellular HUVEC proteoglycans.(A) The core proteins from the chondroitinase-digested proteoglycans from HUVEC-conditioned medium and an equal amount of non–cell-conditioned medium were fractiona... Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Immunostaining of hematopoietic cells with antiserglycin IgY.Cells were prepared as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Immunofluorescent staining of HUVECs with antiserglycin IgY and anti-vWF IgG.Cells were prepared as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Confocal analysis of localization of serglycin, vWF, and tPA in HUVECs.Slides were prepared and examined by confocal microscopy as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology Effect of TNF-α on secretion of serglycin and vWF from HUVECs.Cells were treated as described in the text. Barbara P. Schick et al. Blood 2001;97:449-458 ©2001 by American Society of Hematology
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