Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Establishment of a sandwich ELISA for detecting L 59 LAP-DPs. a Schematic diagram of the ELISA for L 59 LAP-DPs. In the ELISA, L 59 LAP-DPs are first captured by coated L59 antibodies (Ab) and then sandwiched by biotin-conjugated anti-LAP antibodies (αLAP-Ab), which form a complex with strep-AP. For detection, an enzyme substrate is added and absorbance at 405 nm was measured. b , c Incubation time- and PLK concentration-dependent increases in the absorbance of the samples containing LAP incubated with PLK. The LAP (final 25 nM) was digested with 12.5 nM (open triangle), 25 nM (open reverse triangle), and 50 nM (open square) PLK, or without PLK (open circle) for 0–90 min, and then the samples were diluted by 1/50 and subjected to the L 59 LAP-DP ELISA ( b ). Also, 25 nM rhLAP was digested with 50 nM PLK (open square) or PLN (cross mark), or PLK in the presence of 5 μM camostat mesilate (open diamond) ( c ). All data were presented as mean ± SD from two different experiments ​*p-value <0.05, **p-value <0.01, ***p-value<0.001 obtained comparing to corresponding control values

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Correlation among L 59 LAP-DPs and active TGF-β in the culture medium, and intracellular signal transduction. a , c The ×9CAGA-Luc-transformed CCL64 cells were cultured in PLK-added CM derived from HEK293T cells overexpressing hLTGF-β1. After 6 h, the levels of active TGF-β1 and L 59 LAP-DPs were determined by respective ELISAs ( a ), and the extent of TGF-β signaling was measured by luciferase activity in CCL64 cells ( c ). b , d The scatterplots between the levels of L 59 LAP-DPs and active TGF-β shown in a ( b ) and between increases in L 59 LAP-DP levels and increases in luminescence from the values obtained compared to basal levels in the absence of PLK shown in c ( d ). A significant positive correlation was seen ( b ) * p-value <0.05, ***p-value <0.001 obtained comparing to 0

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Transformation Assay, Cell Culture, Derivative Assay, Luciferase, Activity Assay

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques:

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Primers designed for ribozyme synthesis.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Primers for conventional RT-PCR and real-time qPCR.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Verification of ribozyme transgene knockdown of OPG and RANK in MDA-MB-231 cells. Reduced expression of OPG and RANK were confirmed at a transcript level using RT-PCR (A and D) and qPCR (B and E) compared to the control cell line. Western blotting was used to confirm knockdown of OPG and RANK at a protein level (C and F respectively). PCR and western blotting were normalised against GAPDH. Representative images and data shown. ** p≤0.01, *** p≤0.001.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Impact of OPG and RANK knockdown on MDA-MB-231 cell proliferation in vitro . Suppression of OPG expression had no effect on MDA-MB-231 cell proliferation after 5-day incubation compared to MDA-MB-231 pEF6 control cells (A). Reduced RANK expression in MDA-MB-231 cells resulted in a significant decrease in cell proliferation after 5-day incubation compared with control cells (B). Treatment of the MDA-MB-231 pEF6 control cell line with 40 ng/ml HGF, 50 μg/ml BME or a combination of 40 ng/ml HGF and 50 μg/ml BME resulted in a significant increase in cell proliferation after 5-day incubation compared to the untreated cells (C). After 5-day treatment of MDA-MB-231 OPGKD cells or MDA-MB-231 RANKKD cells with 40 ng/ml HGF, 50 μg/ml BME or a combination of 40 ng/ml HGF and 50 μg/ml BME no changes were seen compared to the respective untreated cells (D and E). Data represent the mean of 4 independent repeats, error bars represent SEM. * p≤0.05.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Knockdown, In Vitro, Expressing, Incubation, Control

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Impact of reduced OPG and RANK expression in MDA-MB-231 cells on cell-matrix adhesion in vitro . Reduced OPG expression did not alter MDA-MB-231 cell-matrix adhesion compared with control cells (A). Reduced RANK expression resulted in a significant decrease in MDA-MB-231 cell-matrix adhesion compared with control cells (B). When MDA-MB-231 pEF6 control cells were treated with 40 ng/ml HGF or 50 μg/ml BME or a combination of 40 ng/ml HGF and 50 μg/ml BME did not significantly alter MDA-MB-231 cell-matrix adhesion (C). MDA-MB-231 OPGKD cells treated with 40 ng/ml HGF or 50 μg/ml BME also did not alter cell-matrix adhesion, however, a combined of HGF and BME treatment significantly decreased cell matrix adhesion (D). MDA-MB-231 RANKKD cells treated with 40 ng/ml HGF, 50 μg/ml BME or combined HGF and BME did not affect cell-matrix adhesion compared to the untreated cells (E). Data represent the mean of 4 independent repeats, error bars represent SEM. * p≤0.05.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Expressing, In Vitro, Control

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Effect of OPG and RANK knockdown on MDA-MB-231 cell motility. MDA-MB-231 OPGKD cells showed significantly increased motility compared with MDA-MB-231 pEF6 control cells (A). Reduced RANK expression resulted in a significant decrease in MDA-MB-231 cell motility compared to MDA-MB-231 pEF6 control cells (B). Treatment of MDA-MB-231 pEF6 control cells with 40 ng/ml HGF or 50 μg/ml BME did not significantly alter cell motility. However, a combination of 40 ng/ml HGF and 50 μg/ml BME significantly increased cell motility compared to the untreated control (C). Treatment of MDA-MB-231 OPGKD or MDA-MB-231 RANKKD cells with 40 ng/ml HGF and/or 50 μg/ml BME did not significantly impact cell motility (D and E, respectively). Data represent the mean of 4 independent repeats, error bars represent SEM. * p≤0.05, ** p≤0.01, *** p≤0.001.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Knockdown, Control, Expressing

Journal: International Journal of Oncology

Article Title: Importance of osteoprotegrin and receptor activator of nuclear factor κB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

doi: 10.3892/ijo.2016.3339

Figure Lengend Snippet: Impact of reduced OPG and RANK expression on MDA-MB-231 cell invasion in vitro . MDA-MB-231 OPGKD cells showed significantly increased cell invasion compared with MDA-MB-231 pEF6 control cells (A). MDA-MB-231 RANKKD cells showed reduced cell invasion compared with MDA-MB-231 pEF6 control cells (B). Treatment of MDA-MB-231 pEF6 control cells with 40 ng/ml HGF, 50 μg/ml BME or a combination of 40 ng/ml HGF and 50 μg/ml BME do not impact cell invasion (C). Treatment of MDA-MB-231 OPGKD cells with 40 ng/ml HGF or a combination of 40 ng/ml HGF and 50 μg/ml BME resulted in a significant decrease in cell invasion (D). MDA-MB-231 RANKKD cells treated with 40 ng/ml HGF, 50 μg/ml BME or combined 40 ng/ml HGF and 50 μg/ml BME showed non-significant increases in cell invasion (E). Data represent the mean of 3 independent repeats, error bars represent SEM. * p=≤0.05, ** p≤0.01.

Article Snippet: OPG expression was detected using anti-OPG antibody [R&D Systems, Abingdon, UK (BAF805)], RANK expression was detected using anti-RANK antibody [Santa Cruz Biotechnology, Inc., CA, USA (sc-9072)].

Techniques: Expressing, In Vitro, Control

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Designation of the DNA vaccine.(a) B cell epitope scanning of human DKK1 was performed with the software DNASTAR 7.1. (b) The affinity of epitopes was measured by indirect ELISA. (c) The separated epitopes were immunized BALB/c mice and the immunogenicity of epitopes was measured by sandwich ELISA. (d) The separated epitopes were injected to BALB/c mice for seven days. TRAP staining was performed to identify the mature osteoclasts. Magnification: 200x; data are expressed as the mean ± SEM.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Software, Indirect ELISA, Immunopeptidomics, Sandwich ELISA, Injection, Staining

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Construction of the DNA vaccine. (a) The maps of recombinant DKK1 DNA vaccine. (b) The amino acid sequence of human DKK1. (c) The recombinant amino acid sequence of DKK1 DNA vaccine. Red line, 110–144aa; green line, 153–181aa; orange line, 182–216aa; blue line, 228–253aa; black line, signal peptide; box, PADRE.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Recombinant, Sequencing

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Expression and immunogenicity of the DNA vaccine. (a–c) Expression of the multiepitope protein in vitro and in vivo were determined in cell culture supernatants by ELISA, cell lysates by Western blotting, and the muscles of mice by immunohistochemical analysis. (d-e) The DNA vaccine induced a specific IgG antibody against human DKK1. The titer and the end-point titer of the specific antibody were tested by ELISA. Bars indicate 100 μ m. Data are expressed as the mean ± SEM, * P < 0.05, *** P < 0.001.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Expressing, Immunopeptidomics, In Vitro, In Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Muscles, Immunohistochemical staining

Journal: Molecular Brain

Article Title: Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo ; characterisation, subcellular localisation and modulation of the receptors

doi: 10.1186/s13041-015-0138-6

Figure Lengend Snippet: Schematic of the insulin/IGF1R signalling pathway. A simplified representation of the insulin/IGF1 signalling pathway depicting the downstream activation of Akt and p44-42 MAPK through binding of insulin or IGF1 to their respective receptors. Furthermore insulin can bind to IGF1R and IGF1 to IR and IR α-subunits/ β-subunits can form heterodimers with IGF1R α-subunits /β-subunits adding further complexity at the level of the receptors. There are also numerous downstream feedback loops within this and other signalling pathways which act to regulate signalling through this pathway at multiple levels

Article Snippet: To impair signalling through IGF1R cultures were treated with 11 μg/ml IGF1 receptor monoclonal antibody (24 h) (MAB391, R&D Systems, MN, USA).

Techniques: Activation Assay, Binding Assay

Journal: Molecular Brain

Article Title: Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo ; characterisation, subcellular localisation and modulation of the receptors

doi: 10.1186/s13041-015-0138-6

Figure Lengend Snippet: Insulin/IGF1 pathway characterisation in astrocytes. Immunoblots of astrocytes cultured in either serum containing or serum deprived media with additional supplementation from either a recombinant human 1 μM insulin or b 11.2nM recombinant human IGF1. Respresentative images from blots probed with antibodies against IRβ, IGF1Rβ, IRS1, IRS2, pAkt, Total Akt, p44/42 MAPK are shown. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa)

Article Snippet: To impair signalling through IGF1R cultures were treated with 11 μg/ml IGF1 receptor monoclonal antibody (24 h) (MAB391, R&D Systems, MN, USA).

Techniques: Western Blot, Cell Culture, Recombinant, Control, Molecular Weight

Journal: Molecular Brain

Article Title: Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo ; characterisation, subcellular localisation and modulation of the receptors

doi: 10.1186/s13041-015-0138-6

Figure Lengend Snippet: Impairment of IGF1 signalling using a monoclonal IGF1 antibody (MAB391). Human astrocytes treated with 11 μg/ml MAB391 for 24 h ( a ). Representative immunoblots demonstrate reductions in IGF1Rβ, IRβ and pAkt in response to MAB391 with no impact on total IRS1 or downstream signalling through p44/42 MAPK. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa). b Bar charts show quantification of immunoblots, pAkt was normalised to Akt/α-tubulin. c Immunofluorescence showing the reduction in IGF1R, scale bars represent 10 μM. d qPCR analysis of IGF1Rβ RNA after 24 h show no differences at the RNA level. Data represents mean + SEM ( n = 3, 3 replicates/experiments, Unpaired t -test, ** p < 0.01)

Article Snippet: To impair signalling through IGF1R cultures were treated with 11 μg/ml IGF1 receptor monoclonal antibody (24 h) (MAB391, R&D Systems, MN, USA).

Techniques: Western Blot, Control, Molecular Weight, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 is dynamically expressed in skeletal muscle tissue. (A) Total RNAs were extracted from E14.5 and E19.5 mouse hind limb muscles and further subjected to RT-PCR to detect the mRNA expression of Fzd9 (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). GAPDH expression was used as a loading control gene. The gel is representative of two experiments performed by triplicate. (B) Proteins from mouse hind limb skeletal muscles obtained at different developmental stages (E14.5, E16.5, and E19.5) were fractionated by SDS-PAGE and immunoblotted for Fzd9 and β-actin ( upper panel ). Fzd9 is expressed at the protein level in all the analyzed developmental stages since the expected 56kDa band is detected. A representative gel shows that Fzd9 band intensity displays a progressive decrease from E14.5 to E19.5. Data represent the mean ± SD of Fzd9/β-actin ratio from three experiments, normalized to E14.5 ( lower panel ) ( * p < 0.05, ** p < 0.01, ANOVA using Bonferroni's post-hoc analysis). (C) Whole-mounted diaphragms from E17.5 and P0 were stained with anti Fzd9 antibody (green) together with αBTX to reveal the postsynaptic densities (blue). Fzd9 is abundant in the synaptic domain of embryonic NMJs, where it displays a punctate expression pattern. The insets ( lower panels ) show that some αBTX-positive regions were not labeled with anti Fzd9 antibodies. Pictures are representative of at least three experiments performed by triplicate.

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Muscles, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Reverse Transcription, Control, SDS Page, Staining, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 impairs agrin-dependent AChR clustering in myotubes. (A) Fzd9 is expressed in the muscle cell line C2C12 throughout differentiation. C2C12 cells were cultured in vitro and differentiated for 0, 3, or 6 days (d0-d6). Total proteins were subjected to Western blot analyses. An expected 56 kDa band is gradually increased during C2C12 cells differentiation. α-tubulin expression was used as a loading control. (B) C2C12 myotubes differentiated for 6 days were analyzed by immunocytochemistry to detect Fzd9. Fzd9 is localized to the plasma membrane of the myotubes (green, upper panel ), similar to Glut1, which was used as a marker of plasma membrane (red, middle panel ). The merge image ( lower panel ) reveals the co-localization of Fzd9 and Glut1. (C) Differentiated C2C12 myotubes were subjected to a sequential fractionation procedure to isolate samples enriched in cytoplasm (cyt) or plasma membrane (mb) proteins. Western blot analyzes showed that α-tubulin is specifically detected in cytoplasmic fractions, whereas the vitamin C transporter SVCT2 was only present in membrane-enriched protein fractions ( left panel ). C2C12 myoblasts were transfected either with GFP or Fzd9 and differentiated. Sequential protein lysates were analyzed by Western blot. Both endogenous Fzd9 (GFP-transfected cells) or overexpressed Fzd9 (Fzd9-transfected cells) were found predominantly in the plasma membrane and were absent in the cytoplasm. As a loading control, β-actin is found only in the cytoplasm-enriched fraction ( right panel ). (D) Myoblasts transfected with plasmids coding for GFP (control) or Fzd9 were differentiated into myotubes and subsequently incubated with 200 pM neural agrin. αBTX staining allows the visualization of the AChRs (red). Automatized quantification of aggregates shows that Fzd9 overexpression induces a decrease in the number of AChR clusters per myotube, as well as a reduction in the total area and average size of AChR clusters, compared to controls. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( ** p < 0.01, *** p < 0.001 compared to GFP controls, t -test).

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Cell Culture, In Vitro, Western Blot, Expressing, Control, Immunocytochemistry, Clinical Proteomics, Membrane, Marker, Fractionation, Transfection, Incubation, Staining, Over Expression

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Down-regulation of Fzd9 increases agrin-dependent AChR clustering in myotubes. (A,B) The efficiency of shFzd9 was tested by its ability to impair overexpression/function of the Fzd9 construct. (B) HEK293 cells were transfected with Fzd9HA together with a control shRNA (pFUX) or shFzd9, followed by protein homogenization and Western blot. Whereas a 56 kDa band corresponding to Fzd9 is detected in the membrane-enriched fraction of the control condition, Fzd9 expression is drastically silenced in cells transfected with shFzd9. (B) The efficiency of the shFzd9 to affect the functionality of Fzd9HA was assessed by co-transfecting Fzd9HA and Wnt2 in the presence or absence of the shFzd9 plasmid in HEK293 cells. Activation of the TOPflash luciferase reporter gene was used as a readout of activation of the canonical Wnt pathway. These experiments were performed at least three times by triplicate ( ** p < 0.01, *** p < 0.001, t -test). (C) Myoblasts were transfected either with GFP or shFzd9 and differentiated for 5 days. Myotubes were treated with neural agrin and further stained with an anti-Fzd9 antibody (red), along with αBTX (blue) to detect AChR clusters and DAPI (yellow) to stain nuclei. shFz9-transfected myotubes display silenced Fzd9 expression and an apparent increase in the number of AChR clusters compared to GFP-expressing myotubes. (D) Myoblasts transfected either with GFP or shFzd9 and grown for 5 days were treated with neural agrin and further stained with αBTX to detect AChR clusters (red). Myotubes expressing the GFP protein present in the shFzd9 plasmid show a significant increase in the number of AChR clusters, an increase in the total area of AChR clusters, as well as on the average size of AChR clusters, when compared to control myotubes that only express GFP. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( * p < 0.05, *** p < 0.001 compared to GFP controls, t -test).

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Over Expression, Construct, Transfection, Control, shRNA, Homogenization, Western Blot, Membrane, Expressing, Plasmid Preparation, Activation Assay, Luciferase, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 enhances β-catenin accumulation in myotubes. (A) GFP and Fzd9-transfected myotubes were immunostained with an anti β-catenin antibody (red). Fluorescence intensity of transfected myotubes was quantified using Metamorph. Quantification of the data ( right panel ) shows that the expression of Fzd9 induces a significant ~2-fold accumulation of β-catenin in the sarcoplasma, compared to control GFP-expressing myotubes. (B) Total protein samples from GFP- and Fzd9-transfected myotubes were separated by SDS-PAGE and immunoblotted with Fzd9 and β-catenin antibodies. Quantification of the Fzd9 or β-catenin against β-actin band intensity ratios shows that Fzd9-overexpressing myotubes display a ~2-fold increase in β-catenin cytosolic levels, which is equivalent to the ~2-fold increase observed for Fzd9 levels, compared to control myotubes ( right panel ). Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to control GFP cells; *** p < 0.001, t -test, compared to the GFP group). (C) Whole-mounted diaphragms of E17.5 mice were immunostained to detect Fzd9 (green) and β-catenin (red). AChR aggregates were stained with αBTX (blue). β-catenin is associated to the sarcolemma of embryonic muscle fibers, including the membrane domains where AChR clusters and Fzd9 are localized. Pictures are representative of at least three experiments performed by triplicate.

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Transfection, Fluorescence, Expressing, Control, SDS Page, Staining, Membrane