rabbit polyclonal anti fibulin 5  (Millipore)

Journal: PLoS ONE

Article Title: Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

doi: 10.1371/journal.pone.0115362

Figure Lengend Snippet: ( A ) MPPs displayed differential activity in affecting The pattern of extracellular HtrA1 complexes (arrowheads) from HeLa cell low-serum conditioned medium is differentially altered by treatment with MPPs (25 mM, 37°C, 1 hr), as demonstrated when probed with polyclonal anti-HtrA1 antibody. ( B ) MPPs may induce conformational changes upon binding to HtrA1. The accessibility of N-terminal and C-terminal epitopes in the presence of MPPs was determined by ELISA and compared to DMSO controls. Error bars indicate the standard deviation. ( C ) Extracellular HtrA1 (arrows) could be precipitated from HEK-HtrA1 conditioned medium using HEMIN-conjugated agarose beads but not control, unconjugated agarose beads. ( D ) Competitive binding experiments using conditioned medium from HEK-HtrA1 or HeLa cells pre-incubated with MPPs revealed reduced recovery of HtrA1 (arrows) in the presence of competitor compounds when probed with polyclonal anti-HtrA1 antibody. ( E ) Degradation of Fibulin 5 in fixed HeLa cells treated with HtrA1 conditioned medium was enhanced in the presence of TPP, ZPP and PPP-IX. RMA: Rosmarinic acid, TPP: tin protoporphyrin IX, ZPP: zinc protoporphyrin IX, PPP-IX: protoporphyrin IX, HEM: HEMIN, CPP: cobalt protoporphyrin IX, CM: conditioned medium.

Article Snippet: All antibodies were purchased from Cell Signaling Technology except monoclonal anti-HtrA1 and polyclonal anti-HtrA2 (R&D Systems), rabbit polyclonal anti-Fibulin-5 (Millipore), monoclonal anti-V5 (Invitrogen), and rabbit polyclonal anti-HtrA1 (kind gift from Dr. Sascha Fauser, University of Cologne ).

Techniques: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation

Journal: PLoS ONE

Article Title: Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

doi: 10.1371/journal.pone.0115362

Figure Lengend Snippet: ( A–C ) PPP-IX treatment induced apoptotic cell death in HeLa cells. ( A ) Bright field images of HeLa cells treated with PPP-IX (lower left) revealed increased membrane blebbing (arrows) compared to DMSO treated cells (upper left). Dotted squares indicate the location of magnified views shown in the middle panel. DAPI staining of nuclei (right panel) revealed increased numbers of fragmented nuclei (arrows) in PPP-IX treated cells compared to DMSO treatment. ( B ) PARP and Caspase-3 are cleaved in lysates from HeLa cells undergoing cell death in response to increasing concentrations of PPP-IX. Fibulin-5 cleavage was used to confirm drug efficacy. ( C ) PPP-IX treatment reduced cell viability, as determined by the MTT assay, in a dose-dependent manner. ( D-F ) Knockdown of HtrA1 conferred resistance to PPP-IX induced cell death. ( D ) HtrA1 knockdown reduced the extent of PARP and Caspase-3 cleavage in PPP-IX treated cells. ( E ) The relative activity of Caspase-3/7 compared to that for DMSO treated, siCon transfected cells is shown. Caspase activation is reduced in siHtrA1 transfected HeLa cells undergoing PPP-IX induced cell death. ( F ) The viability of cells transfected with control (siCon) or HtrA1-targeting (siHtrA1) was assessed in the presence and absence of PPP-IX. PPP-IX-induced cell death was attenuated by loss of HtrA1. FL: full length intact protein, CL: cleaved protein.

Article Snippet: All antibodies were purchased from Cell Signaling Technology except monoclonal anti-HtrA1 and polyclonal anti-HtrA2 (R&D Systems), rabbit polyclonal anti-Fibulin-5 (Millipore), monoclonal anti-V5 (Invitrogen), and rabbit polyclonal anti-HtrA1 (kind gift from Dr. Sascha Fauser, University of Cologne ).

Techniques: Staining, MTT Assay, Activity Assay, Transfection, Activation Assay

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Glucokinase activation was associated with upregulation of Fbln5 gene expression in pancreatic islets. ( a ) Fbln5 mRNA expression levels in isolated islets from C57BL/6 J mice incubated for 2, 6, 12 and 24 hours with 30 μmol/L of GKA CpdA or vehicle (DMSO) (n = 4). ( b ) Fbln5 mRNA expression levels in isolated islets from C57BL/6 J mice (n = 4) after 24 hours’ incubation with 2.8, 5.6, 11.1 or 22.2 mmol/L of glucose. ( c ) Immunoblotting for FBLN5 in islets isolated from wild-type or Fbln5 −/− mice. Full-length blots are presented in Supplementary Fig. ( d ) Immunoblotting for FBLN5 in INS-1 cells treated with vehicle, 22.2 mmol/L of glucose, or 30 μmol/L of GKA CpdA for 24 hours (n = 4). Full-length blots are presented in Supplementary Fig. ( e ) Fbln5 mRNA expression levels in islets from 12-week-old Gck +/− mice and their wild-type littermates after incubation with 30 μmol/L of GKA CpdA for 24 hours (n = 4). ( f ) Fbln5 gene expression levels in islets isolated from 12-week old standard chow-fed mice (SC) and 20-week of high-fat diet (HFD)-fed mice incubated with or without 30 μmol/L of GKA CpdA (vehicle; DMSO) for 24 hours (n = 4). ( g ) Fbln5 mRNA expression levels in islets from 12-week-old Irs-2 −/− mice and their wild-type littermates after incubation with 30 μmol/L of GKA CpdA for 24 hours (n = 4). ( h – i ) mRNA expression levels of Fbln5 ( h ) or Ki67 ( i ) in isolated islets from 2-, 6- and 12-week-old C57BL/6 J mice (n = 3–4). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Activation Assay, Expressing, Isolation, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Glucose-signal induced Fbln5 upregulation via the glucokinase/K ATP channel/calcineurin/NFAT signaling pathway in pancreatic islets. ( a – f ) Fbln5 mRNA expression. Islets from C57BL/6 J mice were incubated with 10 nmol/L of D-mannoheptulose (MH) (n = 4) ( a ), 200 μmol/L of diazoxide (n = 4) ( b ), 200 nmol/L of OSI-906 (n = 4) ( c ), 50 μmol/L of nifedipine (ND), 10 μmol/L of FK506 (n = 3) ( d ), 10 μmol/L of harmine (n = 3) ( e ), or a combination of 10 μmol/L of FK506 and 10 μmol/L of harmine (n = 4) ( f ) for 24 hours in the presence or absence of 30 μmol/L of GKA CpdA (vehicle; DMSO). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Expressing, Incubation

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Fbln5 deficiency in mice had no effect on glucose tolerance, glucose-stimulated insulin secretion or β-cell proliferation. ( a ) Blood glucose levels during an oral glucose tolerance test (OGTT) in 8-week-old Fbln5 −/− mice and wild-type mice (1.5 g/kg body weight, n = 11–13). ( b ) Serum insulin levels during the OGTT (n = 11–13). ( c ) Glucose-stimulated insulin secretion in ten isolated islets from 8-week-old Fbln5 −/− mice and wild-type mice (n = 6–12). ( d , e ) β-cell mass in 8-week-old Fbln5 −/− mice and wild-type mice (n = 5). Representative images of the pancreas showing brown staining of insulin ( d ) and the ratio of the β-cell mass relative to the area of the whole pancreas ( e ) (n = 5). The scale bar represents 100 µm in the pancreas images. ( f , g ) EdU incorporation in islets isolated from 8-week-old C57BL/6 J mice. Representative images of pancreatic islets after 48 hours of incubation with EdU in the presence or absence of 30 μmol/L of GKA CpdA ( f ). Insulin is stained red, nuclei are stained blue with 4′, 6-diamidino-2-phenylindole [DAPI], and EdU-positive nuclei are stained green. The white arrows indicate EdU-positive β-cells. The scale bar represents 50 µm. The ratio of EdU-positive β-cells to the total count of insulin-positive β-cells is shown ( g ). More than 3000 cells were counted in each group (n = 2). Data are represented as means ± SEM. * p < 0.05.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Isolation, Staining, Incubation

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Fbln5 overexpression in INS-1 cells enhanced glucose-stimulated insulin secretion, but inhibited β-cell proliferation. ( a ) Fbln5 mRNA levels in INS-1 cells after infection with Ad-GFP or Ad- Fbln5 for 48 hours (n = 2). ( b ) Immunoblotting for FBLN5 in INS-1 cells infected with Ad-GFP or Ad- Fbln5 . Full-length blots are presented in Supplementary Fig. ( c ) Glucose-stimulated insulin secretion by INS-1 cells infected with Ad-GFP or Ad- Fbln5 (n = 6–12). ( d ) EdU incorporation in INS-1 cells infected with Ad-GFP or Ad- Fbln5 for 48 hours. Cells were incubated for 3 hours with 10 µM of EdU after adenovirus infection. left: Representative images of Ad-GFP infected INS-1 cells or Ad- Fbln5 infected INS-1 cells stained for EdU. Ad-GFP infected cells incubated without EdU are shown as control. Insulin is stained red, nuclei are stained blue with DAPI, and EdU-positive nuclei are stained green. The scale bar represents 50 µm. right: The ratio of EdU-positive cells to the total count of insulin-positive cells is shown. More than 6000 cells were counted in each group (n = 4). ( e ) Ki67 mRNA expression in INS-1 cells infected with Ad- Fbln5 or Ad-GFP for 48 hours (n = 4). Data are represented as means ± SEM. * p < 0.05.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Over Expression, Infection, Western Blot, Incubation, Staining, Expressing

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: A schematic representation of glucokinase-mediated Fbln5 expression in pancreatic islets. Fbln5 gene expression was induced by glucokinase activation through ambient high glucose concentrations or GKA in pancreatic islets. Depolarization of the membrane accompanied by the closure of K ATP channels, Ca 2+ influx and calcineurin activation are required for this Fbln5 upregulation. A DYRK1A inhibitor, harmine, enhanced Fbln5 expression in the islets induced by glucokinase activation, possibly via NFAT signaling. DYRK1A; dual-specificity tyrosine phosphorylation-regulated kinase 1 A, GKA; glucokinase activator, K ATP channel; ATP-sensitive potassium channel, NFAT; nuclear factor of activated T cells, VDCC; voltage-dependent Ca 2+ channels.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Expressing, Activation Assay

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Glucokinase activation was associated with upregulation of Fbln5 gene expression in pancreatic islets. ( a ) Fbln5 mRNA expression levels in isolated islets from C57BL/6 J mice incubated for 2, 6, 12 and 24 hours with 30 μmol/L of GKA CpdA or vehicle (DMSO) (n = 4). ( b ) Fbln5 mRNA expression levels in isolated islets from C57BL/6 J mice (n = 4) after 24 hours’ incubation with 2.8, 5.6, 11.1 or 22.2 mmol/L of glucose. ( c ) Immunoblotting for FBLN5 in islets isolated from wild-type or Fbln5 −/− mice. Full-length blots are presented in Supplementary Fig. ( d ) Immunoblotting for FBLN5 in INS-1 cells treated with vehicle, 22.2 mmol/L of glucose, or 30 μmol/L of GKA CpdA for 24 hours (n = 4). Full-length blots are presented in Supplementary Fig. ( e ) Fbln5 mRNA expression levels in islets from 12-week-old Gck +/− mice and their wild-type littermates after incubation with 30 μmol/L of GKA CpdA for 24 hours (n = 4). ( f ) Fbln5 gene expression levels in islets isolated from 12-week old standard chow-fed mice (SC) and 20-week of high-fat diet (HFD)-fed mice incubated with or without 30 μmol/L of GKA CpdA (vehicle; DMSO) for 24 hours (n = 4). ( g ) Fbln5 mRNA expression levels in islets from 12-week-old Irs-2 −/− mice and their wild-type littermates after incubation with 30 μmol/L of GKA CpdA for 24 hours (n = 4). ( h – i ) mRNA expression levels of Fbln5 ( h ) or Ki67 ( i ) in isolated islets from 2-, 6- and 12-week-old C57BL/6 J mice (n = 3–4). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Activation Assay, Expressing, Isolation, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Glucose-signal induced Fbln5 upregulation via the glucokinase/K ATP channel/calcineurin/NFAT signaling pathway in pancreatic islets. ( a – f ) Fbln5 mRNA expression. Islets from C57BL/6 J mice were incubated with 10 nmol/L of D-mannoheptulose (MH) (n = 4) ( a ), 200 μmol/L of diazoxide (n = 4) ( b ), 200 nmol/L of OSI-906 (n = 4) ( c ), 50 μmol/L of nifedipine (ND), 10 μmol/L of FK506 (n = 3) ( d ), 10 μmol/L of harmine (n = 3) ( e ), or a combination of 10 μmol/L of FK506 and 10 μmol/L of harmine (n = 4) ( f ) for 24 hours in the presence or absence of 30 μmol/L of GKA CpdA (vehicle; DMSO). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Expressing, Incubation

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Fbln5 deficiency in mice had no effect on glucose tolerance, glucose-stimulated insulin secretion or β-cell proliferation. ( a ) Blood glucose levels during an oral glucose tolerance test (OGTT) in 8-week-old Fbln5 −/− mice and wild-type mice (1.5 g/kg body weight, n = 11–13). ( b ) Serum insulin levels during the OGTT (n = 11–13). ( c ) Glucose-stimulated insulin secretion in ten isolated islets from 8-week-old Fbln5 −/− mice and wild-type mice (n = 6–12). ( d , e ) β-cell mass in 8-week-old Fbln5 −/− mice and wild-type mice (n = 5). Representative images of the pancreas showing brown staining of insulin ( d ) and the ratio of the β-cell mass relative to the area of the whole pancreas ( e ) (n = 5). The scale bar represents 100 µm in the pancreas images. ( f , g ) EdU incorporation in islets isolated from 8-week-old C57BL/6 J mice. Representative images of pancreatic islets after 48 hours of incubation with EdU in the presence or absence of 30 μmol/L of GKA CpdA ( f ). Insulin is stained red, nuclei are stained blue with 4′, 6-diamidino-2-phenylindole [DAPI], and EdU-positive nuclei are stained green. The white arrows indicate EdU-positive β-cells. The scale bar represents 50 µm. The ratio of EdU-positive β-cells to the total count of insulin-positive β-cells is shown ( g ). More than 3000 cells were counted in each group (n = 2). Data are represented as means ± SEM. * p < 0.05.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Isolation, Staining, Incubation

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: Fbln5 overexpression in INS-1 cells enhanced glucose-stimulated insulin secretion, but inhibited β-cell proliferation. ( a ) Fbln5 mRNA levels in INS-1 cells after infection with Ad-GFP or Ad- Fbln5 for 48 hours (n = 2). ( b ) Immunoblotting for FBLN5 in INS-1 cells infected with Ad-GFP or Ad- Fbln5 . Full-length blots are presented in Supplementary Fig. ( c ) Glucose-stimulated insulin secretion by INS-1 cells infected with Ad-GFP or Ad- Fbln5 (n = 6–12). ( d ) EdU incorporation in INS-1 cells infected with Ad-GFP or Ad- Fbln5 for 48 hours. Cells were incubated for 3 hours with 10 µM of EdU after adenovirus infection. left: Representative images of Ad-GFP infected INS-1 cells or Ad- Fbln5 infected INS-1 cells stained for EdU. Ad-GFP infected cells incubated without EdU are shown as control. Insulin is stained red, nuclei are stained blue with DAPI, and EdU-positive nuclei are stained green. The scale bar represents 50 µm. right: The ratio of EdU-positive cells to the total count of insulin-positive cells is shown. More than 6000 cells were counted in each group (n = 4). ( e ) Ki67 mRNA expression in INS-1 cells infected with Ad- Fbln5 or Ad-GFP for 48 hours (n = 4). Data are represented as means ± SEM. * p < 0.05.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Over Expression, Infection, Western Blot, Incubation, Staining, Expressing

Journal: Scientific Reports

Article Title: Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

doi: 10.1038/s41598-017-02535-0

Figure Lengend Snippet: A schematic representation of glucokinase-mediated Fbln5 expression in pancreatic islets. Fbln5 gene expression was induced by glucokinase activation through ambient high glucose concentrations or GKA in pancreatic islets. Depolarization of the membrane accompanied by the closure of K ATP channels, Ca 2+ influx and calcineurin activation are required for this Fbln5 upregulation. A DYRK1A inhibitor, harmine, enhanced Fbln5 expression in the islets induced by glucokinase activation, possibly via NFAT signaling. DYRK1A; dual-specificity tyrosine phosphorylation-regulated kinase 1 A, GKA; glucokinase activator, K ATP channel; ATP-sensitive potassium channel, NFAT; nuclear factor of activated T cells, VDCC; voltage-dependent Ca 2+ channels.

Article Snippet: The sections or attached islets on coverslips were immunostained with antibody directed against insulin (Santa Cruz Biotechnology), glucagon (Abcam), somatostatin (GeneTex), CD34 (Santa Cruz Biotechnology), or rabbit polyclonal anti-fibulin-5 (BSYN 1923; 1:100) .

Techniques: Expressing, Activation Assay