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Millipore fgf2 neutralizing antibody 05–117
Fgf2 Neutralizing Antibody 05–117, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fgf2+neutralizing+antibody+05%E2%80%93117/pm35491858-355-7-10?v=Millipore
Average 90 stars, based on 1 article reviews

fgf2 neutralizing antibody 05–117 - by Bioz Stars, 2026-06

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( A , B ) The exogenous FGF2 activates VEGFR signaling in GIST T-1 cells via up-regulation of VEGF-A. ( A ) FGF-2 stimulates, whereas anti-FGF2 neutralizing Abs abrogates VEGFR signaling GIST T-1 cells. Cells were treated with FGF2 (100 ng/mL), IM (0.02 µM) alone or in the presence of anti-FGF2 neutralizing Abs (20 µg/mL) for 72 h. Expression of VEGF-A, total and phosphorylated forms of VEGFR, KIT, MAPK, AKT, and STAT1 was assessed by immunoblot analysis. Actin stain was used as a loading control for each sample. ( B ) Concentration of VEGF-A (pg/mL, measured by ELISA) in supernatants of IM-naive (GIST T-1) cells treated with DMSO (control), FGF-2 (100 ng/mL), IM (0.02 µM) alone or in the presence of anti-FGF-2 Abs (20 µg/mL) for 72 h. Data are presented as median ± SD. Significant differences with p < 0.05 (*), p < 0.01 (**) from n ≥ 3 using unpaired Student’s t -test. ( C ) Changes in the relative expression level of mRNA VEGF-A and VEGFR1, 2, and 3 in GIST T-1 after treatment with FGF-2 (100 ng/mL), as determined by quantitative RT-PCR. The amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for internal control. Data are presented as median ± SD. Significant differences with p < 0.05 (*) from n ≥ 3 using unpaired Student’s t -test. The original Western blot figures can be found in .

Journal: Cancers

Article Title: Fibroblast Growth Factor 2 (FGF2) Activates Vascular Endothelial Growth Factor (VEGF) Signaling in Gastrointestinal Stromal Tumors (GIST): An Autocrine Mechanism Contributing to Imatinib Mesylate (IM) Resistance

doi: 10.3390/cancers16173103

Figure Lengend Snippet: ( A , B ) The exogenous FGF2 activates VEGFR signaling in GIST T-1 cells via up-regulation of VEGF-A. ( A ) FGF-2 stimulates, whereas anti-FGF2 neutralizing Abs abrogates VEGFR signaling GIST T-1 cells. Cells were treated with FGF2 (100 ng/mL), IM (0.02 µM) alone or in the presence of anti-FGF2 neutralizing Abs (20 µg/mL) for 72 h. Expression of VEGF-A, total and phosphorylated forms of VEGFR, KIT, MAPK, AKT, and STAT1 was assessed by immunoblot analysis. Actin stain was used as a loading control for each sample. ( B ) Concentration of VEGF-A (pg/mL, measured by ELISA) in supernatants of IM-naive (GIST T-1) cells treated with DMSO (control), FGF-2 (100 ng/mL), IM (0.02 µM) alone or in the presence of anti-FGF-2 Abs (20 µg/mL) for 72 h. Data are presented as median ± SD. Significant differences with p < 0.05 (*), p < 0.01 (**) from n ≥ 3 using unpaired Student’s t -test. ( C ) Changes in the relative expression level of mRNA VEGF-A and VEGFR1, 2, and 3 in GIST T-1 after treatment with FGF-2 (100 ng/mL), as determined by quantitative RT-PCR. The amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for internal control. Data are presented as median ± SD. Significant differences with p < 0.05 (*) from n ≥ 3 using unpaired Student’s t -test. The original Western blot figures can be found in .

Article Snippet: Neutralizing antibody against bFGF (Anti-FGF2/basic FGF #05-117) and human recombinant FGF-2/basic (FGF2 #01-106) were from Merck KGaA (Darmstadt, Germany).

Techniques: Expressing, Western Blot, Staining, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Amplification

Alcohol exposure alters Fgf2 expression in the dorsal striatum (D.striatum), nucleus accumbens (NAc), and dorsal hippocampus (D.hippocampus). A, Alcohol (2.5 g/kg, 20%, i.p.) or saline was administered once, 2 h (Alcohol 2 h), 6 h (Alcohol 6 h), or 24 h (Alcohol 24 h) before brain tissue collection. n = 5–8 per group. B, Alcohol (2.5 g/kg, 20%, i.p.) or saline was administered once a day for 7 d. Brain tissues were collected 2 h (Alcohol 2 h) or 24 h (Alcohol 24 h) after the last alcohol injection. n = 5–10 per group. A, B, Fgf2 mRNA levels were determined by qRT-PCR and normalized to Gapdh, which was not affected by alcohol on its own (Fig. 1-1. Bar graphs indicate mean + SEM. Each brain region was normalized to its saline control. C, Schematic representation of the sampling regions. *p < 0.05 compared with saline control. **p < 0.01 compared with saline control.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: Alcohol exposure alters Fgf2 expression in the dorsal striatum (D.striatum), nucleus accumbens (NAc), and dorsal hippocampus (D.hippocampus). A, Alcohol (2.5 g/kg, 20%, i.p.) or saline was administered once, 2 h (Alcohol 2 h), 6 h (Alcohol 6 h), or 24 h (Alcohol 24 h) before brain tissue collection. n = 5–8 per group. B, Alcohol (2.5 g/kg, 20%, i.p.) or saline was administered once a day for 7 d. Brain tissues were collected 2 h (Alcohol 2 h) or 24 h (Alcohol 24 h) after the last alcohol injection. n = 5–10 per group. A, B, Fgf2 mRNA levels were determined by qRT-PCR and normalized to Gapdh, which was not affected by alcohol on its own (Fig. 1-1. Bar graphs indicate mean + SEM. Each brain region was normalized to its saline control. C, Schematic representation of the sampling regions. *p < 0.05 compared with saline control. **p < 0.01 compared with saline control.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Expressing, Injection, Quantitative RT-PCR, Sampling

Alcohol-induced increase in Fgf2 expression in the dorsal striatum is mediated by dopamine D2-like receptors. Mice were pretreated with the dopamine D2-like receptor antagonist haloperidol (1 mg/kg, i.p.) or vehicle, and 1 h later were treated with alcohol (2.5 g/kg, 20%, i.p.) or saline, once a day for 7 d. The dorsal striatum was collected 2 h after the last alcohol injection. Fgf2 mRNA levels were determined by qRT-PCR and normalized to Gapdh, which was not affected by alcohol on its own (Fig. 1-1. Bar graphs indicate mean + SEM, normalized to the saline + vehicle control group. n = 5–7 per group. *p < 0.05.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: Alcohol-induced increase in Fgf2 expression in the dorsal striatum is mediated by dopamine D2-like receptors. Mice were pretreated with the dopamine D2-like receptor antagonist haloperidol (1 mg/kg, i.p.) or vehicle, and 1 h later were treated with alcohol (2.5 g/kg, 20%, i.p.) or saline, once a day for 7 d. The dorsal striatum was collected 2 h after the last alcohol injection. Fgf2 mRNA levels were determined by qRT-PCR and normalized to Gapdh, which was not affected by alcohol on its own (Fig. 1-1. Bar graphs indicate mean + SEM, normalized to the saline + vehicle control group. n = 5–7 per group. *p < 0.05.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Expressing, Injection, Quantitative RT-PCR

Voluntary alcohol consumption increases Fgf2 expression in the dorsal striatum (D.striatum). A, Experimental timeline scheme. Mice consumed alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 5 weeks. Control mice consumed water only. Tissues were collected immediately at the end of the last 24-h drinking session (Alcohol group) or 24 h after the termination of the last drinking session (Withdrawal group). B, Fgf2 mRNA levels were determined by qRT-PCR, and normalized to Gapdh, which was not affected by alcohol on its own (Fig. 1-1. Bar graphs indicate mean + SEM. Each brain region was normalized to its water control. n = 8 or 9 per group. **p < 0.01 compared with water control.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: Voluntary alcohol consumption increases Fgf2 expression in the dorsal striatum (D.striatum). A, Experimental timeline scheme. Mice consumed alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 5 weeks. Control mice consumed water only. Tissues were collected immediately at the end of the last 24-h drinking session (Alcohol group) or 24 h after the termination of the last drinking session (Withdrawal group). B, Fgf2 mRNA levels were determined by qRT-PCR, and normalized to Gapdh, which was not affected by alcohol on its own (Fig. 1-1. Bar graphs indicate mean + SEM. Each brain region was normalized to its water control. n = 8 or 9 per group. **p < 0.01 compared with water control.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Expressing, Quantitative RT-PCR

Systemic FGF2 administration increases alcohol, but not saccharin, sucrose, or water intake. Mice were trained to consume excessive amounts of 20% alcohol (A–F), saccharin (G, H), or sucrose (I, J) solution in the intermittent access 2-bottle choice paradigm. A, B, Recombinant FGF2 (40 μg/kg, s.c.) or vehicle was injected 1 h before the beginning of the 24 h alcohol-drinking test session. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C–J, Recombinant FGF2 (80 μg/kg; μg/kg, s.c.) or vehicle was injected 1 h before the beginning of the 24 h alcohol (C–F), saccharin (G, H), or sucrose (I, J) drinking test session. C, Amount of alcohol (g/kg) consumed. D, Preference to alcohol. E, Water intake (ml/kg). F, Total fluid intake (alcohol + water; ml/kg). G, Saccharin (0.005% or 0.03%) solution intake (ml/kg). H, Preference for saccharin solution. I, Sucrose (0.2% or 1%) solution intake (ml/kg). J, Preference for sucrose solution. Bar graphs indicate mean + SEM. n = 7–11 per group. *p < 0.05; #p = 0.09.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: Systemic FGF2 administration increases alcohol, but not saccharin, sucrose, or water intake. Mice were trained to consume excessive amounts of 20% alcohol (A–F), saccharin (G, H), or sucrose (I, J) solution in the intermittent access 2-bottle choice paradigm. A, B, Recombinant FGF2 (40 μg/kg, s.c.) or vehicle was injected 1 h before the beginning of the 24 h alcohol-drinking test session. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C–J, Recombinant FGF2 (80 μg/kg; μg/kg, s.c.) or vehicle was injected 1 h before the beginning of the 24 h alcohol (C–F), saccharin (G, H), or sucrose (I, J) drinking test session. C, Amount of alcohol (g/kg) consumed. D, Preference to alcohol. E, Water intake (ml/kg). F, Total fluid intake (alcohol + water; ml/kg). G, Saccharin (0.005% or 0.03%) solution intake (ml/kg). H, Preference for saccharin solution. I, Sucrose (0.2% or 1%) solution intake (ml/kg). J, Preference for sucrose solution. Bar graphs indicate mean + SEM. n = 7–11 per group. *p < 0.05; #p = 0.09.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Recombinant, Injection

FGF2 infusion into the dorsal striatum leads to increased alcohol consumption and preference. Rats were trained to consume alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 7 weeks before cannulation. Recombinant FGF2 (200 ng/0.75 μl per hemisphere) or vehicle was infused into the dorsal striatum, 10 min before the beginning of an alcohol-drinking session. Alcohol and water intake was measured after 30 min, 4 h, and 24 h. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C, Water intake (ml/kg). D, Total fluid intake (alcohol + water; ml/kg). E, Schematic representation of the cannula tip placement in coronal sections (bregma + mm). Bar graphs indicate mean + SEM adjusted for a within-subjects design (Cousineau, 2005). n = 14 per group. *p < 0.05; **p < 0.01.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: FGF2 infusion into the dorsal striatum leads to increased alcohol consumption and preference. Rats were trained to consume alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 7 weeks before cannulation. Recombinant FGF2 (200 ng/0.75 μl per hemisphere) or vehicle was infused into the dorsal striatum, 10 min before the beginning of an alcohol-drinking session. Alcohol and water intake was measured after 30 min, 4 h, and 24 h. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C, Water intake (ml/kg). D, Total fluid intake (alcohol + water; ml/kg). E, Schematic representation of the cannula tip placement in coronal sections (bregma + mm). Bar graphs indicate mean + SEM adjusted for a within-subjects design (Cousineau, 2005). n = 14 per group. *p < 0.05; **p < 0.01.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Recombinant

Voluntary alcohol consumption increases Fgf2 expression in the DMS of mice and rats. A, Experimental timeline scheme. Mice and rats consumed alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm. Control animals consumed water only. Tissues were collected 24 h after the termination of the last drinking session (Withdrawal group). B, C, Fgf2 mRNA levels were determined by qRT-PCR in the DMS and DLS of mice (B) and rats (C), and normalized to Gapdh. Bar graphs indicate mean + SEM. Each brain region was normalized to its water control. D, Schematic representation of the sampling regions. n = 5 or 6 per group. *p < 0.05 compared with water control.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: Voluntary alcohol consumption increases Fgf2 expression in the DMS of mice and rats. A, Experimental timeline scheme. Mice and rats consumed alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm. Control animals consumed water only. Tissues were collected 24 h after the termination of the last drinking session (Withdrawal group). B, C, Fgf2 mRNA levels were determined by qRT-PCR in the DMS and DLS of mice (B) and rats (C), and normalized to Gapdh. Bar graphs indicate mean + SEM. Each brain region was normalized to its water control. D, Schematic representation of the sampling regions. n = 5 or 6 per group. *p < 0.05 compared with water control.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Expressing, Quantitative RT-PCR, Sampling

FGF2 infusion into the DMS increases alcohol consumption and preference. Rats were trained to consume alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 7 weeks before cannulation. Recombinant FGF2 (200 ng/0.75 μl per hemisphere) or vehicle was infused into the DMS 10 min before the beginning of an alcohol-drinking session. Alcohol and water intake was measured at the end of the 24-h drinking session. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C, Water intake (ml/kg). D, Total fluid intake (alcohol + water; ml/kg). E, Schematic representation of the cannula tip placement in coronal sections (bregma + mm). Bar graphs indicate mean + SEM adjusted for a within-subjects design (Cousineau, 2005). n = 8 per group. *p < 0.05; **p < 0.01.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: FGF2 infusion into the DMS increases alcohol consumption and preference. Rats were trained to consume alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 7 weeks before cannulation. Recombinant FGF2 (200 ng/0.75 μl per hemisphere) or vehicle was infused into the DMS 10 min before the beginning of an alcohol-drinking session. Alcohol and water intake was measured at the end of the 24-h drinking session. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C, Water intake (ml/kg). D, Total fluid intake (alcohol + water; ml/kg). E, Schematic representation of the cannula tip placement in coronal sections (bregma + mm). Bar graphs indicate mean + SEM adjusted for a within-subjects design (Cousineau, 2005). n = 8 per group. *p < 0.05; **p < 0.01.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Recombinant

Inhibition of FGF2 activity in the DMS decreases alcohol consumption and preference. Rats were trained to consume alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 7 weeks before cannulation. Neutralizing antibody against FGF2 (750 ng/0.75 μl per hemisphere) or control IgG was infused into the DMS 1 h before the beginning of an alcohol-drinking session. Alcohol and water intake was measured at the end of the 24-h drinking session. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C, Water intake (ml/kg). D, Total fluid intake (alcohol + water; ml/kg). E, Schematic representation of the cannula tip placement in coronal sections (bregma + mm). Bar graphs indicate mean + SEM adjusted for a within-subjects design (Cousineau, 2005). n = 15 per group. *p < 0.05; **p < 0.01.

Journal: The Journal of Neuroscience

Article Title: Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption

doi: 10.1523/JNEUROSCI.0890-17.2017

Figure Lengend Snippet: Inhibition of FGF2 activity in the DMS decreases alcohol consumption and preference. Rats were trained to consume alcohol in the intermittent access to 20% alcohol in 2-bottle choice paradigm for 7 weeks before cannulation. Neutralizing antibody against FGF2 (750 ng/0.75 μl per hemisphere) or control IgG was infused into the DMS 1 h before the beginning of an alcohol-drinking session. Alcohol and water intake was measured at the end of the 24-h drinking session. A, Amount of alcohol (g/kg) consumed. B, Preference for alcohol, calculated as the ratio of the volume of alcohol solution intake/volume of total fluid intake. C, Water intake (ml/kg). D, Total fluid intake (alcohol + water; ml/kg). E, Schematic representation of the cannula tip placement in coronal sections (bregma + mm). Bar graphs indicate mean + SEM adjusted for a within-subjects design (Cousineau, 2005). n = 15 per group. *p < 0.05; **p < 0.01.

Article Snippet: Neutralizing antibody against FGF2 (05-117) and control IgG (12-371) were purchased from Millipore.

Techniques: Inhibition, Activity Assay

(A) Western blots for differentiation markers and graph of neurite analysis using NeuronJ (mean ± SEM) in 5Y cells expressing nontargeted shRNA or shRNA against TβRIII for 96 hours, with or without 10 ng/ml FGF2 treatment (gray bars). Densitometry for NF160 normalized to β-actin is shown as percent control. P < 0.001 for main effect receptor (2-way ANOVA); P < 0.01 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (B) Western blot for differentiation markers in 5Y cells following 96 hours of adenoviral transduction with GFP control, TβRIII-GFP, TβRIII-HA, or mutant TβRIII-HA lacking GAG chain attachment sites (TβRIII-ΔGAG). Differentiation markers in SHEP cells following 72-hour TβRIII knockdown and rescue. Densitometry for NF160 normalized to β-actin is shown as percent control. NTC, nontargeted control. (C) Western blots in 5Y cells for differentiation markers and phosphorylated and total Erk following 96 hours of transduction and treatment with 1 ng/ml FGF2 (gray bars). Quantification of 3 independent experiments (mean ± SEM). P < 0.0001 for main effect receptor (2-way ANOVA); P < 0.001 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (D) I125 FGF2 binding and crosslinking with total cell lysate (TCL) and TβRIII pull-down (TβRIII IP) in 5Y and SHEP cell lines. Arrows for total cell lysate mark FGFR1 (145 kDa) and TβRIII (80 kDa). Dose course of I125 FGF2 (1 ng/ml, 5 ng/ml, 10 ng/ml); dose course of cold FGF2 (50 ng/ml, 100 ng/ml, 500 ng/ml); GFP condition treated with 10 ng/ml I125 FGF2. Densitometry for TβRIII normalized to β-actin is shown as percent control. (E) Coimmunoprecipitation of TβRIII-HA and FGFR1-FLAG in SHEP cells. PAS beads were used as control.

Journal: The Journal of Clinical Investigation

Article Title: Type III TGF-? receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

doi: 10.1172/JCI69657

Figure Lengend Snippet: (A) Western blots for differentiation markers and graph of neurite analysis using NeuronJ (mean ± SEM) in 5Y cells expressing nontargeted shRNA or shRNA against TβRIII for 96 hours, with or without 10 ng/ml FGF2 treatment (gray bars). Densitometry for NF160 normalized to β-actin is shown as percent control. P < 0.001 for main effect receptor (2-way ANOVA); P < 0.01 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (B) Western blot for differentiation markers in 5Y cells following 96 hours of adenoviral transduction with GFP control, TβRIII-GFP, TβRIII-HA, or mutant TβRIII-HA lacking GAG chain attachment sites (TβRIII-ΔGAG). Differentiation markers in SHEP cells following 72-hour TβRIII knockdown and rescue. Densitometry for NF160 normalized to β-actin is shown as percent control. NTC, nontargeted control. (C) Western blots in 5Y cells for differentiation markers and phosphorylated and total Erk following 96 hours of transduction and treatment with 1 ng/ml FGF2 (gray bars). Quantification of 3 independent experiments (mean ± SEM). P < 0.0001 for main effect receptor (2-way ANOVA); P < 0.001 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (D) I125 FGF2 binding and crosslinking with total cell lysate (TCL) and TβRIII pull-down (TβRIII IP) in 5Y and SHEP cell lines. Arrows for total cell lysate mark FGFR1 (145 kDa) and TβRIII (80 kDa). Dose course of I125 FGF2 (1 ng/ml, 5 ng/ml, 10 ng/ml); dose course of cold FGF2 (50 ng/ml, 100 ng/ml, 500 ng/ml); GFP condition treated with 10 ng/ml I125 FGF2. Densitometry for TβRIII normalized to β-actin is shown as percent control. (E) Coimmunoprecipitation of TβRIII-HA and FGFR1-FLAG in SHEP cells. PAS beads were used as control.

Article Snippet: The neutralizing FGF2 antibody (catalog no. 05-117) was purchased from Millipore and used at a concentration of 5 μg/ml per manufacturer’s instructions.

Techniques: Western Blot, Expressing, shRNA, Transduction, Mutagenesis, Binding Assay

Cells were treated with doses of 10 ng/ml FGF2, 1 μM PD-173074, and 10 μM U0126. (A) Western blot for phosphorylated and total Erk. Differentiation markers after 72-hour TβRIII knockdown and rescue with nontargeted shRNA or shRNA against TβRIII, with or without 1 ng/ml FGF2 treatment (gray bars). Densitometry for pErk normalized to total Erk is shown as percent control. 5Y cells were transduced for 96 hours. Quantification of densitometry from 4 independent experiments is shown (normalized mean ± SEM). P < 0.001 for main effect receptor (2-way ANOVA); P < 0.0001 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (B) Western blots following 96 hours of TβRIII transduction and treatment. Densitometry for NF160 normalized to β-actin is shown as percent control. (C) Western blots following 96 hours of transduction with TβRIII or GFP control and dominant-negative FGFR1 (dnFGFR1) or IRES-GFP vector control. GFP fluorescence was used to verify construct expression. Densitometry for NF160 normalized to β-actin is shown as percent control.

Journal: The Journal of Clinical Investigation

Article Title: Type III TGF-? receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

doi: 10.1172/JCI69657

Figure Lengend Snippet: Cells were treated with doses of 10 ng/ml FGF2, 1 μM PD-173074, and 10 μM U0126. (A) Western blot for phosphorylated and total Erk. Differentiation markers after 72-hour TβRIII knockdown and rescue with nontargeted shRNA or shRNA against TβRIII, with or without 1 ng/ml FGF2 treatment (gray bars). Densitometry for pErk normalized to total Erk is shown as percent control. 5Y cells were transduced for 96 hours. Quantification of densitometry from 4 independent experiments is shown (normalized mean ± SEM). P < 0.001 for main effect receptor (2-way ANOVA); P < 0.0001 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (B) Western blots following 96 hours of TβRIII transduction and treatment. Densitometry for NF160 normalized to β-actin is shown as percent control. (C) Western blots following 96 hours of transduction with TβRIII or GFP control and dominant-negative FGFR1 (dnFGFR1) or IRES-GFP vector control. GFP fluorescence was used to verify construct expression. Densitometry for NF160 normalized to β-actin is shown as percent control.

Article Snippet: The neutralizing FGF2 antibody (catalog no. 05-117) was purchased from Millipore and used at a concentration of 5 μg/ml per manufacturer’s instructions.

Techniques: Western Blot, shRNA, Transduction, Dominant Negative Mutation, Plasmid Preparation, Fluorescence, Construct, Expressing

Cells were treated with doses of 10 ng/ml FGF2, 1 μM PD-173074, and 10 μM U0126. (A) Western blot for Id1 in stable SHEP cells serum-starved 24 hours prior to FGF2 treatment. Densitometry analysis for Id1 normalized to β-actin is shown as percent control. (B) Western blot for Id1 in SHEP cells transduced and treated with FGF2 for 72 hours. Densitometry for Id1 normalized to β-actin is shown as percent control. (C) Western blot for Id1 in 5Y cells transduced for 96 hours. Densitometry for Id1 normalized to β-actin is shown as percent control. (D) 5Y cells were transduced for 96 hours with TβRIII or GFP control and Id1 siRNA (siId1) or nontargeted control siRNA. Densitometry for NF160 normalized to β-actin is shown as percent control. (E) Microarray data set expression of ID1 in tumors with low (bottom 10%) and high (top 10%) TGFBR3 expression (median [horizontal bars] and interquartile range [boxes]). ****P < 0.0001 (Mann-Whitney). Linear regression analysis of ID1 expression, which was dependent on TGFBR3 expression, in the microarray data set.

Journal: The Journal of Clinical Investigation

Article Title: Type III TGF-? receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

doi: 10.1172/JCI69657

Figure Lengend Snippet: Cells were treated with doses of 10 ng/ml FGF2, 1 μM PD-173074, and 10 μM U0126. (A) Western blot for Id1 in stable SHEP cells serum-starved 24 hours prior to FGF2 treatment. Densitometry analysis for Id1 normalized to β-actin is shown as percent control. (B) Western blot for Id1 in SHEP cells transduced and treated with FGF2 for 72 hours. Densitometry for Id1 normalized to β-actin is shown as percent control. (C) Western blot for Id1 in 5Y cells transduced for 96 hours. Densitometry for Id1 normalized to β-actin is shown as percent control. (D) 5Y cells were transduced for 96 hours with TβRIII or GFP control and Id1 siRNA (siId1) or nontargeted control siRNA. Densitometry for NF160 normalized to β-actin is shown as percent control. (E) Microarray data set expression of ID1 in tumors with low (bottom 10%) and high (top 10%) TGFBR3 expression (median [horizontal bars] and interquartile range [boxes]). ****P < 0.0001 (Mann-Whitney). Linear regression analysis of ID1 expression, which was dependent on TGFBR3 expression, in the microarray data set.

Article Snippet: The neutralizing FGF2 antibody (catalog no. 05-117) was purchased from Millipore and used at a concentration of 5 μg/ml per manufacturer’s instructions.

Techniques: Western Blot, Microarray, Expressing, MANN-WHITNEY

5Y, SHEP, and SK-N-AS cells selected for stable expression of TβRIII, TβRIII-ΔGAG, empty vector control (EV), shRNA to TβRIII (shTβRIII), or nontargeted shRNA control (shNTC). (A) Proliferation index from 3 replicates (mean ± SEM) of thymidine incorporation, normalized to empty vector or nontargeted shRNA control lines. P < 0.01 (ANOVA); *P < 0.05 (1-sample t test and 2-tailed Student’s t test). (B) Western blot for p21 in stable cell lines, with or without FGF2 treatment (1 ng/ml for 5Y, 10 ng/ml for SHEP). Densitometry for p21 normalized to β-actin is shown as percent control. (C) 5Y stable orthotopic xenografts (13 mice per group). Tumor weights (mean ± SEM) and images (scale bar in cm) after 7 weeks of growth. Different symbol colors represent different cohorts. P < 0.0001 (1-way ANOVA); pairwise comparisons P < 0.0001 EV vs. TβRIII, P < 0.05 EV vs. TβRIII-ΔGAG (Mann-Whitney) Western blots of tumor lysates. Average NF160 densitometry from 3 replicates normalized to β-actin is shown as percent control. **P < 0.01 (1-sample t test). H&E staining of tumors from each group. T, tumor; A, host adrenal cells. Scale bar: 50 μM. (D) SK-N-AS stable orthotopic xenografts. Tumor images after 4 weeks of growth (scale bar in cm). Western blot of tumor lysates for differentiation markers. (E) Tumor weights at 4 weeks (mean ± SEM). Different symbol colors represent different cohorts. *P < 0.05 (Mann-Whitney). (F) Kaplan-Meier survival analysis (10 mice per group). (G) H&E-stained contralateral adrenal glands from mice at 4 weeks (scale bar: 50 μM). Photograph of macroscopic metastasis to the contralateral adrenal gland at the 4-week end point (scale bar in cm).

Journal: The Journal of Clinical Investigation

Article Title: Type III TGF-? receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

doi: 10.1172/JCI69657

Figure Lengend Snippet: 5Y, SHEP, and SK-N-AS cells selected for stable expression of TβRIII, TβRIII-ΔGAG, empty vector control (EV), shRNA to TβRIII (shTβRIII), or nontargeted shRNA control (shNTC). (A) Proliferation index from 3 replicates (mean ± SEM) of thymidine incorporation, normalized to empty vector or nontargeted shRNA control lines. P < 0.01 (ANOVA); *P < 0.05 (1-sample t test and 2-tailed Student’s t test). (B) Western blot for p21 in stable cell lines, with or without FGF2 treatment (1 ng/ml for 5Y, 10 ng/ml for SHEP). Densitometry for p21 normalized to β-actin is shown as percent control. (C) 5Y stable orthotopic xenografts (13 mice per group). Tumor weights (mean ± SEM) and images (scale bar in cm) after 7 weeks of growth. Different symbol colors represent different cohorts. P < 0.0001 (1-way ANOVA); pairwise comparisons P < 0.0001 EV vs. TβRIII, P < 0.05 EV vs. TβRIII-ΔGAG (Mann-Whitney) Western blots of tumor lysates. Average NF160 densitometry from 3 replicates normalized to β-actin is shown as percent control. **P < 0.01 (1-sample t test). H&E staining of tumors from each group. T, tumor; A, host adrenal cells. Scale bar: 50 μM. (D) SK-N-AS stable orthotopic xenografts. Tumor images after 4 weeks of growth (scale bar in cm). Western blot of tumor lysates for differentiation markers. (E) Tumor weights at 4 weeks (mean ± SEM). Different symbol colors represent different cohorts. *P < 0.05 (Mann-Whitney). (F) Kaplan-Meier survival analysis (10 mice per group). (G) H&E-stained contralateral adrenal glands from mice at 4 weeks (scale bar: 50 μM). Photograph of macroscopic metastasis to the contralateral adrenal gland at the 4-week end point (scale bar in cm).

Article Snippet: The neutralizing FGF2 antibody (catalog no. 05-117) was purchased from Millipore and used at a concentration of 5 μg/ml per manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, shRNA, Western Blot, Stable Transfection, MANN-WHITNEY, Staining

A. bFGF expression was analyzed in CAV1 knocked-down models showing that bFGF expression was affected constantly in all models. The graph shows the quantification of the OD volume determined from the intensity of the bands from the RT-PCR images showing the statistically significant reduction in bFGF in CAV1 knocked-down cells; bars, SD (*P≤0.05 n = 3) B. Immunoblot showing bFGF reduction in the TC71 model either in total protein extract or CM. Actin blot and ponceau staining was used as loading control. Quantitative data measuring relative intensity of the bands is indicated below each lane (n = 3) C. Quantification of endothelial cell migration. CM was used as chemoattractant for endothelial cells. A neutralizing antibody -N. Ab- (2.5 µg/ml and 5 µg/ml) and a recombinant protein -R. Prot- (10 ng/ml); bars, SD (*P<0.002– when compared with parental CM – and *P = 0.0005– when compared with clone n = 3) were used to block and promote migration respectively. RPMI media alone was used as negative control.

Journal: PLoS ONE

Article Title: EphA2-Induced Angiogenesis in Ewing Sarcoma Cells Works through bFGF Production and Is Dependent on Caveolin-1

doi: 10.1371/journal.pone.0071449

Figure Lengend Snippet: A. bFGF expression was analyzed in CAV1 knocked-down models showing that bFGF expression was affected constantly in all models. The graph shows the quantification of the OD volume determined from the intensity of the bands from the RT-PCR images showing the statistically significant reduction in bFGF in CAV1 knocked-down cells; bars, SD (*P≤0.05 n = 3) B. Immunoblot showing bFGF reduction in the TC71 model either in total protein extract or CM. Actin blot and ponceau staining was used as loading control. Quantitative data measuring relative intensity of the bands is indicated below each lane (n = 3) C. Quantification of endothelial cell migration. CM was used as chemoattractant for endothelial cells. A neutralizing antibody -N. Ab- (2.5 µg/ml and 5 µg/ml) and a recombinant protein -R. Prot- (10 ng/ml); bars, SD (*P<0.002– when compared with parental CM – and *P = 0.0005– when compared with clone n = 3) were used to block and promote migration respectively. RPMI media alone was used as negative control.

Article Snippet: Neutralizing antibody against bFGF (Anti-FGF2/basic FGF #05-117) and human recombinant FGF-2/basic (FGF2 #01-106) were from Millipore and were used at mentioned concentrations.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Control, Migration, Recombinant, Blocking Assay, Negative Control

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