Journal: PLoS ONE

Article Title: Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

doi: 10.1371/journal.pone.0031564

Figure Lengend Snippet: ( A ) S-nitrosylation of EMMPRIN was detected by the biotin-switch assay in bovine aortic endothelial cells (BAECs). Input refers to EMMPRIN levels in lysates before performing the biotin-switch assay. Specificity of biotinylation was confirmed by incubating samples in the presence or absence of 2 mM ascorbic acid (AA) during the biotin-switch assay. Arrows indicate S-nitrosylated EMMPRIN (EMP). Shown are representative results from 3 independent experiments. ( B ) S-nitrosylated EMMPRIN levels in BAECs in the basal (non-stimulated, left panel) and after eNOS stimulation with 10 µM of a calcium ionophore (A23187) for 30 min to promote NO production (middle panel). Arrow indicates S-nitrosylated EMMPRIN. BAEC lysates immunoprecipitated (IP) with EMMPRIN (EMP, left panel) or S-nitroso-cysteine (SNO-cys, middle panel) antibodies were blotted with SNO-cys or EMMPRIN antibodies, respectively. The bar graph on the right panel shows band intensities of S-nitrosylated EMMPRIN (arrow in the middle panel) in BAECs stimulated with or without A23187. The blots are representative images from 3 independent experiments. ( C ) S-nitrosylation of GOLPH3 was detected by the biotin-switch assay in BAECs. Input refers to GOLPH3 levels in lysates before performing the biotin-switch assay. Specificity of biotinylation was confirmed by incubating samples in the presence or absence of 2 mM ascorbic acid (AA) during the biotin-switch assay. Arrows indicate S-nitrosylated GOLPH3. The blots are representative images from 3 independent experiments. ( D ) S-nitrosylated GOLPH3 levels in BAECs after eNOS stimulation with 10 µM of A23187 for 30 min to promote NO production (left panel). BAEC lysates immunoprecipitated with S-nitrosocysteine (SNO-cys) antibodies were blotted for GOLPH3. The graph on the right panel shows band intensities of S-nitrosylated GOLPH3 (arrow in the left panel) in BAECs stimulated with or without A23187. Shown are representative results from 3 independent experiments.

Article Snippet: An HA-tagged human GOLPH3 in pBABE-Puro vector was obtained from Addgene (Cambridge, MA).

Techniques: Biotin Switch Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

doi: 10.1371/journal.pone.0031564

Figure Lengend Snippet: ( A ) Immunolabeling of EMMPRIN, eNOS, GOLPH3 and GM130 (a Golgi marker) in bovine aortic endothelial cells (BAECs). The upper panel shows EMMPRIN (red, left) and eNOS (green, center) and their merged image with DNA in blue (right). Arrows indicate where EMMPRIN and eNOS are located. The lower panel shows GOLPH3 (red, left), GM130 (green, center), and their merged image with nucleus in blue (right). Arrows indicate where GOLPH3 and GM130 are located. Scale bar; 10 µm. Images were taken using a Nikon E800 Microscope with a Plan-Fluorchromat 40×/0.75 objective (Nikon, Melville, NY). Shown are representative images from at least 3 independent experiments. ( B ) Immunolabeling of COS cells that were transfected with wild-type eNOS (blue, upper left panel), YFP-EMMPRIN (green, upper right panel) and HA-tagged GOLPH3 (red, lower left panel). The lower right panel shows their merged image. Scale bar; 10 µm. Arrows indicate eNOS-, EMMPRIN- and GOLPH3-rich areas. Scale bar; 10 µm. Shown are representative images from at least 3 independent transfection experiments. ( C ) Immunoprecipitation (IP) using anti-EMMPRIN (EMP) and total goat IgG from BAEC lysates. IP samples were blotted with eNOS and GOLPH3 antibodies. Input refers to eNOS levels in lysates before IP, indicating an equal amount of proteins in lysates used for IP. The blots are representative images from three independent experiments.

Article Snippet: An HA-tagged human GOLPH3 in pBABE-Puro vector was obtained from Addgene (Cambridge, MA).

Techniques: Immunolabeling, Marker, Microscopy, Transfection, Immunoprecipitation