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fgfr1 ab (R&D Systems)

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Rating: 93 (10 reviews)

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R&D Systems fgfr1 ab
Fgfr1 Ab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Figure 1. FGFR1 as an immune adjuvant to combine with ICI in HNSCC mouse models. (a) FGFR1 expression in mouse HNSCC cell lines (MOC1) was examined by Western blotting. (b) Experimental schema. C57BL/6 mice were intradermally injected with MOC1 (1x106). PD173074 (20 mg/kg) and anti-PD-1 Ab (200 μg/mice) was administered 3 times per week from day 18 (tumor size: 7–8 mm). (c) Tumor growth curves. Control (Red), anti-PD-1 Ab monotherapy (Blue), PD173074 monotherapy (Yellow), and combination therapy with PD173074 and anti-PD-1 Ab (Green) (n = 4 or 5 /group). Bars and error bars indicate the mean and SD, respectively (*p < .05, **p < .01, ***<0.001, one-way ANOVA). (d) The mice were sacrificed on day 42, and the percentages of CD4+ T cells and CD8+ T cells in TILs were evaluated with flow cytometry. (e) A representative image of MHC-class I or MHC-class II expression in immunohistochemistry on tumor (Day 42). MHC-class I (central) and MHC-class II (right) was enhanced by PD173074. H&E staining was shown in the left. Scale bars represent 100 µm. (f, g) MHC-class I and MHC-class II expression on MOC1 incubated with 3 μM FGFR-TKIs for 48 hr were evaluated by flow cytometry. MOC1 was treated with or without 50 U/ml IFN-γ for 48 hr before the assay. Red: isotype control, Green: untreated tumor cell lines, Blue: treated with PD173074. Pink: treated with AZD4547. Orange: treated with Erdafitinib. (f) Representative data of flow cytometry. (g) Averages values of mean fluorescence intensity (MFI) by FGFR-TKIs. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Journal: OncoImmunology

Article Title: Immunomodulation via FGFR inhibition augments FGFR1 targeting T-cell based antitumor immunotherapy for head and neck squamous cell carcinoma

doi: 10.1080/2162402x.2021.2021619

Figure Lengend Snippet: Figure 1. FGFR1 as an immune adjuvant to combine with ICI in HNSCC mouse models. (a) FGFR1 expression in mouse HNSCC cell lines (MOC1) was examined by Western blotting. (b) Experimental schema. C57BL/6 mice were intradermally injected with MOC1 (1x106). PD173074 (20 mg/kg) and anti-PD-1 Ab (200 μg/mice) was administered 3 times per week from day 18 (tumor size: 7–8 mm). (c) Tumor growth curves. Control (Red), anti-PD-1 Ab monotherapy (Blue), PD173074 monotherapy (Yellow), and combination therapy with PD173074 and anti-PD-1 Ab (Green) (n = 4 or 5 /group). Bars and error bars indicate the mean and SD, respectively (*p < .05, **p < .01, ***<0.001, one-way ANOVA). (d) The mice were sacrificed on day 42, and the percentages of CD4+ T cells and CD8+ T cells in TILs were evaluated with flow cytometry. (e) A representative image of MHC-class I or MHC-class II expression in immunohistochemistry on tumor (Day 42). MHC-class I (central) and MHC-class II (right) was enhanced by PD173074. H&E staining was shown in the left. Scale bars represent 100 µm. (f, g) MHC-class I and MHC-class II expression on MOC1 incubated with 3 μM FGFR-TKIs for 48 hr were evaluated by flow cytometry. MOC1 was treated with or without 50 U/ml IFN-γ for 48 hr before the assay. Red: isotype control, Green: untreated tumor cell lines, Blue: treated with PD173074. Pink: treated with AZD4547. Orange: treated with Erdafitinib. (f) Representative data of flow cytometry. (g) Averages values of mean fluorescence intensity (MFI) by FGFR-TKIs. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Article Snippet: The membrane was incubated with mouse anti-human FGFR1 Abs (M19B2, Novus Biologicals) and mouse anti-human βactin Abs (C4, Santa Cruz Biotechnology, Santa Cruz, CA) and detected via chemiluminescence using the Amersham ECL Prime Western blotting Detection System (GE Healthcare Life Sciences) and Invitrogen iBright Imaging Systems 1500 (Invitrogen, Thermo Fisher Scientific).

Techniques: Adjuvant, Expressing, Western Blot, Injection, Control, Flow Cytometry, Immunohistochemistry, Staining, Incubation, Fluorescence

Figure 2. The changes of HLA and CIITA expression on HNSCC cell lines by FGFR1-TKIs. (a) FGFR1 expression in human HNSCC cell lines was examined by Western blotting. Jurkat (leukemia cells) was used as a negative control. (b, c) HLA-class I and HLA-DR and expression on HNSCC cell lines incubated with 3 μM FGFR-TKIs for 48 hr were evaluated by flow cytometry. HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. Green: isotype control, Red: untreated tumor cell lines, Blue: treated with PD173074. Pink: treated with AZD4547. Orange: treated with Erdafitinib. (b) Representative data of flow cytometry. (c) Averages values of mean fluorescence intensity (MFI). (d-g) FGFR-TKIs (3 μM) upregulated CIITA expression in HNSCC cell lines. HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. (d) Representative data of Western blotting without IFN-γ. (e) Quantitative analysis of protein expression. (f) Representative data of Western blotting with IFN-γ. (g) Quantitative analysis of protein expression. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Journal: OncoImmunology

Article Title: Immunomodulation via FGFR inhibition augments FGFR1 targeting T-cell based antitumor immunotherapy for head and neck squamous cell carcinoma

doi: 10.1080/2162402x.2021.2021619

Figure Lengend Snippet: Figure 2. The changes of HLA and CIITA expression on HNSCC cell lines by FGFR1-TKIs. (a) FGFR1 expression in human HNSCC cell lines was examined by Western blotting. Jurkat (leukemia cells) was used as a negative control. (b, c) HLA-class I and HLA-DR and expression on HNSCC cell lines incubated with 3 μM FGFR-TKIs for 48 hr were evaluated by flow cytometry. HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. Green: isotype control, Red: untreated tumor cell lines, Blue: treated with PD173074. Pink: treated with AZD4547. Orange: treated with Erdafitinib. (b) Representative data of flow cytometry. (c) Averages values of mean fluorescence intensity (MFI). (d-g) FGFR-TKIs (3 μM) upregulated CIITA expression in HNSCC cell lines. HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. (d) Representative data of Western blotting without IFN-γ. (e) Quantitative analysis of protein expression. (f) Representative data of Western blotting with IFN-γ. (g) Quantitative analysis of protein expression. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Techniques: Expressing, Western Blot, Negative Control, Incubation, Flow Cytometry, Control, Fluorescence

Figure 5. Direct killing of FGFR1 expressing HNSCC cells by FGFR1305-319-reactiveCD4+ T cell lines. (a) FGFR1305-319-reactive CD4+ T cell lines were co-cultured with HLA-DR matched or unmatched HNSCC cell lines expressing FGFR1 for 48 hr. K1 and K3 were restricted to HLA-DR4, and K2 was restricted to HLA-DR53. The cell lines used were HSC2 (HLA-DR13), HSC3 (HLA-DR15), HSC4 (HLA-DR1,4, and 53), Sa-3 (HLA-DR9, 10, and 53), and HPC-92Y (HLA-DR4, 9, and 53). HNSCC cell lines were treated with 500 U/ml IFN-γ for 48 hr before the assay. IFN-γ production in the supernatants was evaluated by ELISA. (b) Granzyme-B production from FGFR1305-319-reactive CD4+ T cell line (K1: HLA-DR4 restricted) was assessed in supernatants co-cultured with HLA-DR matched or unmatched HNSCC cell lines. (c) Killing activity of FGFR1305-319-reactive CD4+ T cell line (K1) was evaluated by co-culturing with CSFE-labeled HNSCC cell lines for 6 hr with various E: T (Effector: Target cells) ratio, and measuring percentages of CFSE+ 7-AAD+ dead cells with flow cytometry. (d) Representative data of flow cytometry in the killing assay (Effector to target ratio was 20:1). Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Journal: OncoImmunology

Article Title: Immunomodulation via FGFR inhibition augments FGFR1 targeting T-cell based antitumor immunotherapy for head and neck squamous cell carcinoma

doi: 10.1080/2162402x.2021.2021619

Figure Lengend Snippet: Figure 5. Direct killing of FGFR1 expressing HNSCC cells by FGFR1305-319-reactiveCD4+ T cell lines. (a) FGFR1305-319-reactive CD4+ T cell lines were co-cultured with HLA-DR matched or unmatched HNSCC cell lines expressing FGFR1 for 48 hr. K1 and K3 were restricted to HLA-DR4, and K2 was restricted to HLA-DR53. The cell lines used were HSC2 (HLA-DR13), HSC3 (HLA-DR15), HSC4 (HLA-DR1,4, and 53), Sa-3 (HLA-DR9, 10, and 53), and HPC-92Y (HLA-DR4, 9, and 53). HNSCC cell lines were treated with 500 U/ml IFN-γ for 48 hr before the assay. IFN-γ production in the supernatants was evaluated by ELISA. (b) Granzyme-B production from FGFR1305-319-reactive CD4+ T cell line (K1: HLA-DR4 restricted) was assessed in supernatants co-cultured with HLA-DR matched or unmatched HNSCC cell lines. (c) Killing activity of FGFR1305-319-reactive CD4+ T cell line (K1) was evaluated by co-culturing with CSFE-labeled HNSCC cell lines for 6 hr with various E: T (Effector: Target cells) ratio, and measuring percentages of CFSE+ 7-AAD+ dead cells with flow cytometry. (d) Representative data of flow cytometry in the killing assay (Effector to target ratio was 20:1). Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Flow Cytometry

Figure 6. The existence of FGFR1-reactive precursor T cells in HNSCC patients. (a) PBMCs from HNSCC patients were co-cultured with FGFR1305-319 peptides for 2 cycles every one week. T cell response to FGFR305-319 peptide was assessed by measuring IFN-γ production in the supernatants using ELISA. Anti-HLA-DR mAb was used to assess HLA restriction of the T cells. PADRE peptide was used as a positive control. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test). (b) The clinical characteristics and peptide-reactivity of the 6 HNSCC patients. <: less than the lower limit of detection.

Journal: OncoImmunology

Article Title: Immunomodulation via FGFR inhibition augments FGFR1 targeting T-cell based antitumor immunotherapy for head and neck squamous cell carcinoma

doi: 10.1080/2162402x.2021.2021619

Figure Lengend Snippet: Figure 6. The existence of FGFR1-reactive precursor T cells in HNSCC patients. (a) PBMCs from HNSCC patients were co-cultured with FGFR1305-319 peptides for 2 cycles every one week. T cell response to FGFR305-319 peptide was assessed by measuring IFN-γ production in the supernatants using ELISA. Anti-HLA-DR mAb was used to assess HLA restriction of the T cells. PADRE peptide was used as a positive control. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test). (b) The clinical characteristics and peptide-reactivity of the 6 HNSCC patients. <: less than the lower limit of detection.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Positive Control

R1 and IMC-H7 MAbs binding properties. A , overview of FGFR1 homodimer structure and relevant epitopes. The extracellular domain of FGFR1 contains three Ig domains (D1-D3). An existing crystal structure of the D2D3 domains of FGFR1 bound to FGF2 (PDB 1CVS ) provides a more detailed view of the binding sites of FGF2 to FGFR1 and homodimerization interface of FGFR1 mediated through the D2 domains. One copy of FGFR1 is shown in dark blue ribbons and the other copy is shown as a gray surface . The two FGF2 proteins are colored in orange . The epitope of R1MAb1/2 ( red ) as determined by peptide ELISAs is colored in red and overlaps predominantly with the FGFR1 homodimerization interface . B , bio-layer interferometry (BLI)-based epitope binning demonstrates that R1MAb1, R1MAb2, and IMC-H7 bind to overlapping epitopes. R1MAb2 was captured on the surface and saturated with FGFR1 protein. R1FAb1 or IMC-H7 FAb were added. Neither the R1FAb1 or IMC-H7 FAb could bind to FGFR1 in the presence of R1MAb2. C , binding inhibition curves of anti-FGFR1 R1MAb1 ( blue circle ), R1MAb2 ( red circle ), and IMC-H7 MAb ( black circle ) in the presence of Iodine-125 FGF2 on COS7 cells expressing FGFR1c. Data are representative of two independent experiments are performed in triplicates. Data are means ± S.D. FGFR1, Fibroblast growth factor receptor 1.

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: R1 and IMC-H7 MAbs binding properties. A , overview of FGFR1 homodimer structure and relevant epitopes. The extracellular domain of FGFR1 contains three Ig domains (D1-D3). An existing crystal structure of the D2D3 domains of FGFR1 bound to FGF2 (PDB 1CVS ) provides a more detailed view of the binding sites of FGF2 to FGFR1 and homodimerization interface of FGFR1 mediated through the D2 domains. One copy of FGFR1 is shown in dark blue ribbons and the other copy is shown as a gray surface . The two FGF2 proteins are colored in orange . The epitope of R1MAb1/2 ( red ) as determined by peptide ELISAs is colored in red and overlaps predominantly with the FGFR1 homodimerization interface . B , bio-layer interferometry (BLI)-based epitope binning demonstrates that R1MAb1, R1MAb2, and IMC-H7 bind to overlapping epitopes. R1MAb2 was captured on the surface and saturated with FGFR1 protein. R1FAb1 or IMC-H7 FAb were added. Neither the R1FAb1 or IMC-H7 FAb could bind to FGFR1 in the presence of R1MAb2. C , binding inhibition curves of anti-FGFR1 R1MAb1 ( blue circle ), R1MAb2 ( red circle ), and IMC-H7 MAb ( black circle ) in the presence of Iodine-125 FGF2 on COS7 cells expressing FGFR1c. Data are representative of two independent experiments are performed in triplicates. Data are means ± S.D. FGFR1, Fibroblast growth factor receptor 1.

Article Snippet: After a 24 h incubation, cells were cultured with FGF ligand and/or FGFR1 Abs diluted in DiscoverX-recommended medium for 22 h. Following stimulation, signal was detected using the PathHunter Detection Kit according to the recommended protocol.

Techniques: Binding Assay, Inhibition, Expressing

Impact of R1 and IMC-H7 MAbs and FAbs on FGFR1-dependent signaling in the presence of FGF1 or FGF2. A , schematic representation of FGFR1 signaling pathways activated in response to FGF1/2 binding. Measurement of ( B ) FRS2 phosphorylation in COS7 cells, ( C ) Elk1 activity in COS7 cells, and ( D ) PLCγ1 recruitment in U2OS cells expressing FGFR1c and incubated in the presence of a constant amount of FGF2 and increasing amounts of R1 and IMC-H7 MAbs or FAbs. For ( B – D ), data are normalized to the maximum signal obtained in the absence of Abs. Data are means ± S.D. of at least three independent experiments performed in triplicate. E , immunoblots performed on lysates prepared from CAL-120 cells incubated in the presence of 0.3 nM FGF1 and 0.08 to 80 nM of FP-1039, 1 to 1000 nM of AZD4547, or 0.1 to 100 nM of R1MAb2 or IMC-H7 MAb. Immunoblots are representative of three independent experiments. The quantitation data from these three independent experiments are shown in <xref ref-type=

Fig. S2 . F , CAL-120 cells 7-days proliferation assay in the absence or presence of FGF1 (0.3 nM) and R1MAb2, IMC-H7 MAb, FGF ligand trap (FP-1039), or AZD4547. Data are normalized as follows: 100% = 0.3 nM FGF1 and 0% = no FGF1. Data are means ± S.D and are representative of three independent experiments. FGF, fibroblast growth factor; FGFR1, FGF receptor 1. " width="100%" height="100%">

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Impact of R1 and IMC-H7 MAbs and FAbs on FGFR1-dependent signaling in the presence of FGF1 or FGF2. A , schematic representation of FGFR1 signaling pathways activated in response to FGF1/2 binding. Measurement of ( B ) FRS2 phosphorylation in COS7 cells, ( C ) Elk1 activity in COS7 cells, and ( D ) PLCγ1 recruitment in U2OS cells expressing FGFR1c and incubated in the presence of a constant amount of FGF2 and increasing amounts of R1 and IMC-H7 MAbs or FAbs. For ( B – D ), data are normalized to the maximum signal obtained in the absence of Abs. Data are means ± S.D. of at least three independent experiments performed in triplicate. E , immunoblots performed on lysates prepared from CAL-120 cells incubated in the presence of 0.3 nM FGF1 and 0.08 to 80 nM of FP-1039, 1 to 1000 nM of AZD4547, or 0.1 to 100 nM of R1MAb2 or IMC-H7 MAb. Immunoblots are representative of three independent experiments. The quantitation data from these three independent experiments are shown in Fig. S2 . F , CAL-120 cells 7-days proliferation assay in the absence or presence of FGF1 (0.3 nM) and R1MAb2, IMC-H7 MAb, FGF ligand trap (FP-1039), or AZD4547. Data are normalized as follows: 100% = 0.3 nM FGF1 and 0% = no FGF1. Data are means ± S.D and are representative of three independent experiments. FGF, fibroblast growth factor; FGFR1, FGF receptor 1.

Techniques: Protein-Protein interactions, Binding Assay, Phospho-proteomics, Activity Assay, Expressing, Incubation, Western Blot, Quantitation Assay, Proliferation Assay

Impact of R1 and IMC-H7 MAbs and FAbs on FGFR1-dependent signaling in the absence of FGF ligands. A , measurement of FRS2 phosphorylation in COS7 cells expressing FGFR1c and incubated in the presence of increasing amounts of R1 and IMC-H7 MAbs, FAbs, or FGF2. Data are normalized to the maximum signal obtained with FGF2. Data are means ± S.D. of at least three independent experiments performed in triplicate. B , MAPK target gene transcript levels in CAL-120 cells upon treatment with R1MAb2 (1 μg/ml), IMC-H7 MAb (1 μg/ml), or FGFR small molecule inhibitor AZD4547 (0.2 μM) for 8 h. C , 7-days proliferation assay using CAL-120 cells treated with R1MAb2, R1FAb2, IMC-H7 MAb, IMC-H7 FAb, or FGF1 at indicated concentrations. D , representative images of cells treated with increasing amounts of R1MAb2 at day 0 and day 7 posttreatment. The scale bar represents 300 μm. B - D , data are means ± S.D. and representative of three independent experiments, performed in triplicates. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; MAPK, mitogen-activated protein kinase.

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Impact of R1 and IMC-H7 MAbs and FAbs on FGFR1-dependent signaling in the absence of FGF ligands. A , measurement of FRS2 phosphorylation in COS7 cells expressing FGFR1c and incubated in the presence of increasing amounts of R1 and IMC-H7 MAbs, FAbs, or FGF2. Data are normalized to the maximum signal obtained with FGF2. Data are means ± S.D. of at least three independent experiments performed in triplicate. B , MAPK target gene transcript levels in CAL-120 cells upon treatment with R1MAb2 (1 μg/ml), IMC-H7 MAb (1 μg/ml), or FGFR small molecule inhibitor AZD4547 (0.2 μM) for 8 h. C , 7-days proliferation assay using CAL-120 cells treated with R1MAb2, R1FAb2, IMC-H7 MAb, IMC-H7 FAb, or FGF1 at indicated concentrations. D , representative images of cells treated with increasing amounts of R1MAb2 at day 0 and day 7 posttreatment. The scale bar represents 300 μm. B - D , data are means ± S.D. and representative of three independent experiments, performed in triplicates. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; MAPK, mitogen-activated protein kinase.

Techniques: Phospho-proteomics, Expressing, Incubation, Proliferation Assay

Assess ment of differences in FGFR1 signaling pathways triggered by R1MAb2 or IMC-H7 MAb versus FGF ligands. A , cell-surface SNAP-tagged FGFR1c interaction determined using TR-FRET in the presence of buffer or isotype controls ( blue histobars ), FGF2 ( purple histobar ), or R1MAb2, FAb2, IMC-H7 MAb, and FAb ( black histobars ). Results are normalized to the TR-FRET signal obtained in the absence of stimulation. B , Western blot analysis performed using lysates prepared from FGFR1-amplified CAL-120 cells incubated with a concentration range of FGF1 starting at 1 nM or a concentration range of R1MAb2 or IMC-H7 MAb starting at 10 nM. Immunoblots are representative of three independent experiments. The quantitation data from these three independent experiments are shown in <xref ref-type=

Fig. S5 . C , PLCγ1 recruitment measured in U2OS cells expressing ProLink tagged FGFR1c and an Enzyme Acceptor tagged SH2 domain in the presence of increasing amounts of R1MAb2, IMC-H7 MAb, or FGF2. D , phosphorylation status of cancer-related kinases assessed using phospho-kinase arrays in CAL-120 cells treated with FGF1/heparin or R1MAb2. Array Part A contains 29 Abs printed in duplicate, and array Part B contains 16 Abs printed in duplicate. All arrays were processed at the same time. E , immunoblots and quantitation data of FGFR1 downstream signaling molecules performed with lysates from CAL-120 cells transfected with either a nontargeting control (NTC) or PLCγ1 siRNA and treated with FGF1 or R1MAb2 48 h posttransfection. The quantitation data are means ± S.D. of three independent experiments. Student’s t -tests for 2 comparisons: ∗ p < 0.05. F , calcium release signal measured upon stimulation of FGFR1c-expressing HEK293 cells with increasing amounts of R1MAb2, IMC-H7 MAb, or FGF2. For (C and F), data are normalized to the maximum signal obtained with FGF2. For ( A , C , and F ) data are means ± S.D. of at least three independent experiments performed in triplicate. One-way ANOVA with Tukey’s multiple comparison test: ∗∗∗∗ p < 0.0001. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; TR-FRET, time-resolved FRET. " width="100%" height="100%">

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Assess ment of differences in FGFR1 signaling pathways triggered by R1MAb2 or IMC-H7 MAb versus FGF ligands. A , cell-surface SNAP-tagged FGFR1c interaction determined using TR-FRET in the presence of buffer or isotype controls ( blue histobars ), FGF2 ( purple histobar ), or R1MAb2, FAb2, IMC-H7 MAb, and FAb ( black histobars ). Results are normalized to the TR-FRET signal obtained in the absence of stimulation. B , Western blot analysis performed using lysates prepared from FGFR1-amplified CAL-120 cells incubated with a concentration range of FGF1 starting at 1 nM or a concentration range of R1MAb2 or IMC-H7 MAb starting at 10 nM. Immunoblots are representative of three independent experiments. The quantitation data from these three independent experiments are shown in Fig. S5 . C , PLCγ1 recruitment measured in U2OS cells expressing ProLink tagged FGFR1c and an Enzyme Acceptor tagged SH2 domain in the presence of increasing amounts of R1MAb2, IMC-H7 MAb, or FGF2. D , phosphorylation status of cancer-related kinases assessed using phospho-kinase arrays in CAL-120 cells treated with FGF1/heparin or R1MAb2. Array Part A contains 29 Abs printed in duplicate, and array Part B contains 16 Abs printed in duplicate. All arrays were processed at the same time. E , immunoblots and quantitation data of FGFR1 downstream signaling molecules performed with lysates from CAL-120 cells transfected with either a nontargeting control (NTC) or PLCγ1 siRNA and treated with FGF1 or R1MAb2 48 h posttransfection. The quantitation data are means ± S.D. of three independent experiments. Student’s t -tests for 2 comparisons: ∗ p < 0.05. F , calcium release signal measured upon stimulation of FGFR1c-expressing HEK293 cells with increasing amounts of R1MAb2, IMC-H7 MAb, or FGF2. For (C and F), data are normalized to the maximum signal obtained with FGF2. For ( A , C , and F ) data are means ± S.D. of at least three independent experiments performed in triplicate. One-way ANOVA with Tukey’s multiple comparison test: ∗∗∗∗ p < 0.0001. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; TR-FRET, time-resolved FRET.

Techniques: Protein-Protein interactions, Western Blot, Amplification, Incubation, Concentration Assay, Quantitation Assay, Expressing, Phospho-proteomics, Transfection, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Assessment of phospho-tyrosine profile differences in CAL-120 treated with R1MAb2 or FGF1. A , illustration of experimental workflow of phospho-tyrosine affinity enrichment. B , volcano plots representing the changes in peptide abundance between control and FGF1- ( left ) or R1MAb2-treated ( right ) samples. Peptides with significant (Linear mix-effects model: p < 0.05) changes greater than 2-fold are plotted in red . C and D , comparison of changes for pTyr sites abundance in FGF1-treated samples against R1MAb2-treated samples. Plots highlight sites more abundant upon (C) both treatments or (D) FGF1 treatment only. Colors reflect proteins of interest, and shape shows the significance of the change in abundance. FGFR1, Fibroblast growth factor receptor 1; pTyr, phospho-tyrosine.

Techniques: Control, Comparison

Impact of FGFR1 MAbs and AZD4547 on weight loss in C.B-17 SCID mice inoculated with CAL-120 cells. Body weight changes of C.B-17 SCID mice bearing similar sized tumors after administration of vehicle, AZD4547, IMC-H7 MAb, or R1MAb2 were monitored. Grouped analysis and individual curves (n = 9 per group) are shown. FGFR1, Fibroblast growth factor receptor 1.

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Impact of FGFR1 MAbs and AZD4547 on weight loss in C.B-17 SCID mice inoculated with CAL-120 cells. Body weight changes of C.B-17 SCID mice bearing similar sized tumors after administration of vehicle, AZD4547, IMC-H7 MAb, or R1MAb2 were monitored. Grouped analysis and individual curves (n = 9 per group) are shown. FGFR1, Fibroblast growth factor receptor 1.

Techniques:

Proposed model of FGFR1 activation in the presence of FGF ligand, FGFR1 Ab alone (R1MAb or IMC-H7 MAb), or FGF ligand and FGFR1 MAb. In the presence of FGF, FGFR forms stable dimers with FGF contacting both receptors and both FGFR1 extracellular domains interacting through a small patch in D2. A conformational change occurs at the TM level that translates into full activation of the FRS2- and PLCγ1-dependent signaling pathways. In the presence of the FGFR1 MAb, the ECDs are further apart and cannot interact through D2, which may result in a KD configuration favorable to phosphorylation events supporting FRS2-dependent signaling only and cell proliferation. When both MAb and FGF ligand are present, the MAb indirectly competes with FGF binding, and at high MAb concentration, a similar phenotype as previously described with MAb alone is observed. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; KD, kinase domain.

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Proposed model of FGFR1 activation in the presence of FGF ligand, FGFR1 Ab alone (R1MAb or IMC-H7 MAb), or FGF ligand and FGFR1 MAb. In the presence of FGF, FGFR forms stable dimers with FGF contacting both receptors and both FGFR1 extracellular domains interacting through a small patch in D2. A conformational change occurs at the TM level that translates into full activation of the FRS2- and PLCγ1-dependent signaling pathways. In the presence of the FGFR1 MAb, the ECDs are further apart and cannot interact through D2, which may result in a KD configuration favorable to phosphorylation events supporting FRS2-dependent signaling only and cell proliferation. When both MAb and FGF ligand are present, the MAb indirectly competes with FGF binding, and at high MAb concentration, a similar phenotype as previously described with MAb alone is observed. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; KD, kinase domain.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Binding Assay, Concentration Assay

( A ) FGFR1-EGFP was transfected into PC12 cells. Twenty-four hours after transfection, cultures were treated with 50 ng/ml NGF and LMB (100 ng/ml in 0.1% v/v ethanol) or ethanol alone (0.1% v/v ethanol) for an additional 48 h and during subsequent imaging. Examples of FGFR1-EGFP expressing cells before and after photo-bleaching are shown. DIC image indicates the nuclear and cytoplasmic regions. About 1/3 nuclear area of PC12 cell was bleached by high intensity laser and 2–3 regions of interest (ROI) intensity were measured. ( B ) Single-exponential analysis of FGFR1-EGFP FLIP regression in cytoplasm and nucleus showed that the diffusion rate of FGFR1-EGFP is affected by NGF treatment in live cells. Individual curves represent means of at least 23 cells. NGF significantly increases the FGFR1-EGFP exchange between nucleus and cytoplasm (half-time decreases) without affecting the FGFR1-EGFP mobile population. ( C ) Single-exponential analysis of FGFR1-EGFP FLIP regression in the cytoplasm shows that NGF facilitates FGFR1-EGFP trafficking between the cytoplasm and nucleus (half-time decreases from 121.5 sec to 89.7 sec; One-way AVOVA, LSD*p<0.001 different to -LMB/−NGF). LMB (100 ng/ml) alone markedly reduces the FGFR1-EGFP half-time (*p<0.001 different to -LMB/−NGF). However, no additive effect of NGF and LMB was observed (p = 0.76), indicating that NGF accelerates FGFR1 nuclear accumulation by reducing nuclear export.

Journal: PLoS ONE

Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

doi: 10.1371/journal.pone.0068931

Figure Lengend Snippet: ( A ) FGFR1-EGFP was transfected into PC12 cells. Twenty-four hours after transfection, cultures were treated with 50 ng/ml NGF and LMB (100 ng/ml in 0.1% v/v ethanol) or ethanol alone (0.1% v/v ethanol) for an additional 48 h and during subsequent imaging. Examples of FGFR1-EGFP expressing cells before and after photo-bleaching are shown. DIC image indicates the nuclear and cytoplasmic regions. About 1/3 nuclear area of PC12 cell was bleached by high intensity laser and 2–3 regions of interest (ROI) intensity were measured. ( B ) Single-exponential analysis of FGFR1-EGFP FLIP regression in cytoplasm and nucleus showed that the diffusion rate of FGFR1-EGFP is affected by NGF treatment in live cells. Individual curves represent means of at least 23 cells. NGF significantly increases the FGFR1-EGFP exchange between nucleus and cytoplasm (half-time decreases) without affecting the FGFR1-EGFP mobile population. ( C ) Single-exponential analysis of FGFR1-EGFP FLIP regression in the cytoplasm shows that NGF facilitates FGFR1-EGFP trafficking between the cytoplasm and nucleus (half-time decreases from 121.5 sec to 89.7 sec; One-way AVOVA, LSD*p<0.001 different to -LMB/−NGF). LMB (100 ng/ml) alone markedly reduces the FGFR1-EGFP half-time (*p<0.001 different to -LMB/−NGF). However, no additive effect of NGF and LMB was observed (p = 0.76), indicating that NGF accelerates FGFR1 nuclear accumulation by reducing nuclear export.

Article Snippet: Nuclear accumulation of FGFR1 was detected using monoclonal N-terminal FGFR1 Ab from Abcam ( ) as well the polyclonal C-terminal FGFR1 Ab from Santa Cruz ( , supplementary material) and the N-terminal FGFR1mcAB6 ( supplementary material), giving further support to the observations of nuclear accumulation of full length FGFR1 .Importantly, the nuclear accumulation of truncated FGFR1 (approximately 50 kDa) was demonstrated in breast cancer cells .

Techniques: Transfection, Imaging, Expressing, Diffusion-based Assay

( A ) PC12 cells were maintained in RPMI1640 with 1% horse serum in the presence or absence of NGF (50 ng/ml) for 7 days. The cytoplasmic and nuclear proteins were prepared for electrophoresis (45 µg protein/lane) and immunoblotting with monoclonal N-terminal FGFR1 antibody (Abcam). Cytoplasmic and nuclear immunoreactive FGFR1 protein bands of 140, 110, 100 and 90 kDa correspond to different degrees of FGFR1 glycosylation . Normalization to GADPH and matrin-3 verifies cytoplasmic depletion and nuclear accumulation of FGFR1, respectively. ( B ) PC12 cells were treated with 50 ng/ml NGF for the durations indicated or maintained without NGF. The effect of NGF on FGFR1 expression and nuclear accumulation is illustrated by immunostaining with monoclonal N-terminal FGFR1 antibody plus goat-anti mouse Alexa488. ( C ) Human neuroblastoma cells were incubated with or without 100 ng/ml NGF for 1, 2 or 7 days and immunolabeled with N-terminal FGFR1 antibody plus goat-anti mouse Alexa488. Nuclear DNA was stained with DAPI. Nuclear accumulation of FGFR1 is observed after NGF treatment. Bar length - 10 µm (B and C).

Journal: PLoS ONE

Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

doi: 10.1371/journal.pone.0068931

Figure Lengend Snippet: ( A ) PC12 cells were maintained in RPMI1640 with 1% horse serum in the presence or absence of NGF (50 ng/ml) for 7 days. The cytoplasmic and nuclear proteins were prepared for electrophoresis (45 µg protein/lane) and immunoblotting with monoclonal N-terminal FGFR1 antibody (Abcam). Cytoplasmic and nuclear immunoreactive FGFR1 protein bands of 140, 110, 100 and 90 kDa correspond to different degrees of FGFR1 glycosylation . Normalization to GADPH and matrin-3 verifies cytoplasmic depletion and nuclear accumulation of FGFR1, respectively. ( B ) PC12 cells were treated with 50 ng/ml NGF for the durations indicated or maintained without NGF. The effect of NGF on FGFR1 expression and nuclear accumulation is illustrated by immunostaining with monoclonal N-terminal FGFR1 antibody plus goat-anti mouse Alexa488. ( C ) Human neuroblastoma cells were incubated with or without 100 ng/ml NGF for 1, 2 or 7 days and immunolabeled with N-terminal FGFR1 antibody plus goat-anti mouse Alexa488. Nuclear DNA was stained with DAPI. Nuclear accumulation of FGFR1 is observed after NGF treatment. Bar length - 10 µm (B and C).

Techniques: Electrophoresis, Western Blot, Glycoproteomics, Expressing, Immunostaining, Incubation, Immunolabeling, Staining

( A ) PC12 cells were transfected with two plasmids, one expressing recombinant FGFR1 or control pcDNA3.1 and the second expressing EGFP. EGFP diffuses throughout the cell permitting visualization of the entire neuritic network. More than 90% of cells co-expressed transfected plasmids as reported in previous studies , . Twenty-four hours after transfection cultures were switched to 1% horse serum medium with or without (control) 50 ng/ml NGF for an additional 10 days, after which the cells were imaged using fluorescent microscopy. Bar length - 100 µm. ( B ) The longest process in an individual transfected cell was measured using ImageJ .* mark comparison to pCDNA3.1 (-NGF) and # to pCNA3.1 (+NGF). Transfection of constitutive nuclear FGFR1(SP−/NLS) increased neurite outgrowth approximately 3-fold (*p<0.001, One-Way ANOVA, LSD). A similar increase was induced by NGF treatment of pcDNA3.1 transfected cells (*p<0.001). Cells transfected with dominant negative FGFR1(SP−/NLS)(TK-) or FGFR1(TK-) display no significant changes in average neurite length in the absence or presence of NGF. ( C ) PC12 cells were transfected with a th- Luciferase reported plasmid and dominant negative FGFR1(TK-), FGFR1(SP−/NLS)(TK-) or control pcDNA3.1(-). 24 hours after transfection cells were treated for an additional 24 hours with indicated concentrations of NGF. The th -Luc expression in the presence of NGF was significantly reduced by both FGFR1(TK-) and FGFR1(SP−/NLS)(TK-). Dominant negative FGFR1 constructs had no significant effect on basal promoter activity. One-Way ANOVA, LSD: * (p<0.01) - mark comparison to (-NGF) within individual plasmid transfection groups; # (p<0.05) - comparison to pCDNA3.1+3 ng/ml NGF; •(p<0.01) - comparison to pcDNA3.1+10 ng/ml NGF; and x (p<0.001) - comparison to pCNA3.1+30 ng/ml NGF. Two-Way ANOVA shows interactions (p<0.001) between NGF and plasmid constructs (control, FGFR1(TK-) or FGFR1(SP−/NLS)(TK-)). ( D ) PC12 cells were transfected with th -Luc and control pcDNA3.1(-) or pcDNA3.1 expressing an active constitutive nuclear FGFR1(SP−/NLS). 24 hours after transfection cells were treated for an additional 6 or 8 hours with NGF. FGFR1(SP−/NLS) increases th promoter activity 2-fold in the absence of NGF but shows no additive stimulation in the presence of NGF. One-Way ANOVA, LSD: * ( p<0.001) - comparison to (-NGF) within individual plasmid transfection groups; # (p<0.05) - comparison to pCDNA3.1 (- NGF).

Journal: PLoS ONE

Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

doi: 10.1371/journal.pone.0068931

Figure Lengend Snippet: ( A ) PC12 cells were transfected with two plasmids, one expressing recombinant FGFR1 or control pcDNA3.1 and the second expressing EGFP. EGFP diffuses throughout the cell permitting visualization of the entire neuritic network. More than 90% of cells co-expressed transfected plasmids as reported in previous studies , . Twenty-four hours after transfection cultures were switched to 1% horse serum medium with or without (control) 50 ng/ml NGF for an additional 10 days, after which the cells were imaged using fluorescent microscopy. Bar length - 100 µm. ( B ) The longest process in an individual transfected cell was measured using ImageJ .* mark comparison to pCDNA3.1 (-NGF) and # to pCNA3.1 (+NGF). Transfection of constitutive nuclear FGFR1(SP−/NLS) increased neurite outgrowth approximately 3-fold (*p<0.001, One-Way ANOVA, LSD). A similar increase was induced by NGF treatment of pcDNA3.1 transfected cells (*p<0.001). Cells transfected with dominant negative FGFR1(SP−/NLS)(TK-) or FGFR1(TK-) display no significant changes in average neurite length in the absence or presence of NGF. ( C ) PC12 cells were transfected with a th- Luciferase reported plasmid and dominant negative FGFR1(TK-), FGFR1(SP−/NLS)(TK-) or control pcDNA3.1(-). 24 hours after transfection cells were treated for an additional 24 hours with indicated concentrations of NGF. The th -Luc expression in the presence of NGF was significantly reduced by both FGFR1(TK-) and FGFR1(SP−/NLS)(TK-). Dominant negative FGFR1 constructs had no significant effect on basal promoter activity. One-Way ANOVA, LSD: * (p<0.01) - mark comparison to (-NGF) within individual plasmid transfection groups; # (p<0.05) - comparison to pCDNA3.1+3 ng/ml NGF; •(p<0.01) - comparison to pcDNA3.1+10 ng/ml NGF; and x (p<0.001) - comparison to pCNA3.1+30 ng/ml NGF. Two-Way ANOVA shows interactions (p<0.001) between NGF and plasmid constructs (control, FGFR1(TK-) or FGFR1(SP−/NLS)(TK-)). ( D ) PC12 cells were transfected with th -Luc and control pcDNA3.1(-) or pcDNA3.1 expressing an active constitutive nuclear FGFR1(SP−/NLS). 24 hours after transfection cells were treated for an additional 6 or 8 hours with NGF. FGFR1(SP−/NLS) increases th promoter activity 2-fold in the absence of NGF but shows no additive stimulation in the presence of NGF. One-Way ANOVA, LSD: * ( p<0.001) - comparison to (-NGF) within individual plasmid transfection groups; # (p<0.05) - comparison to pCDNA3.1 (- NGF).

Techniques: Transfection, Expressing, Recombinant, Control, Microscopy, Comparison, Dominant Negative Mutation, Luciferase, Plasmid Preparation, Construct, Activity Assay

( A )PC12 cells were incubated with NGF (50 ng/ml) or without NGF for 4 hours. mRNA levels were measured using RT-qPCR relative to the house keeping gene cyclophilin A mRNA. Representative experiment shows NGF-induced up-regulation of fgfr1, fgf2, dcx, th, βIII-tubulin, nurr1 and nur77 mRNAs. Similar results were observed in three experiments. ( B ) PC12 cells were transfected with recombinant FGFR1 or control β-gal and with EGFP to mark transfected cells. Images show examples of EGFP fluorescence (left) and DCX-IR (right) in the same β-gal transfected, NGF treated cells. Bars represent average DCX-IR intensity (±SEM) in the nuclei of least 12 EGFP+ cells measured using imageJ. Treatment with NGF for 7 days leads to a gradual outgrowth of neurites with intensified DCX-IR. Cells transfected with FGFR1(SP−/NLS)(TK-) and treated with NGF display significantly less DCX-IR (One-Way ANOVA, LSD *p<0.05 different to –NGF within individual plasmid groups; X p<0.001 different from β-gal+ NGF. Two-Way ANOVA (p<0.001) shows an interaction between transfected plasmid DNA and NGF. Bar length - 20 µm. ( C ) PC12 cells were transfected with FGFR1(TK-), FGFR1(SP−/NLS)(TK-) or β-gal. After 24 hr, cells were incubated for an additional 24 hours with or without NGF, and subsequently with 0.1 mM 5′-flurouridine (FU) for 25 minutes to label newly synthesized RNA. FU-RNA was detected using anti-BrdU and confocal microscopy. Bars represent average (±SEM) intensity of nuclear 5′-FU-IR in at least 19 EGFP expressing cells. NGF significantly increased FU-IR intensity in PC12 cells. Dominant negative FGFR1 significantly decreased the FU-IR intensity in NGF treated and non-treated cells. One-Way ANOVA, LSD: *p<0.05 difference between – NGF and+NGF within individual plasmid groups; X p<0.05 difference from β-gal -NGF; #p<0.05 difference from β-gal +NGF. Two-Way ANOVA (p = 0.057) suggests a potential interaction between transfected plasmid DNA and NGF. Bar length - 10 µm.

Journal: PLoS ONE

Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

doi: 10.1371/journal.pone.0068931

Figure Lengend Snippet: ( A )PC12 cells were incubated with NGF (50 ng/ml) or without NGF for 4 hours. mRNA levels were measured using RT-qPCR relative to the house keeping gene cyclophilin A mRNA. Representative experiment shows NGF-induced up-regulation of fgfr1, fgf2, dcx, th, βIII-tubulin, nurr1 and nur77 mRNAs. Similar results were observed in three experiments. ( B ) PC12 cells were transfected with recombinant FGFR1 or control β-gal and with EGFP to mark transfected cells. Images show examples of EGFP fluorescence (left) and DCX-IR (right) in the same β-gal transfected, NGF treated cells. Bars represent average DCX-IR intensity (±SEM) in the nuclei of least 12 EGFP+ cells measured using imageJ. Treatment with NGF for 7 days leads to a gradual outgrowth of neurites with intensified DCX-IR. Cells transfected with FGFR1(SP−/NLS)(TK-) and treated with NGF display significantly less DCX-IR (One-Way ANOVA, LSD *p<0.05 different to –NGF within individual plasmid groups; X p<0.001 different from β-gal+ NGF. Two-Way ANOVA (p<0.001) shows an interaction between transfected plasmid DNA and NGF. Bar length - 20 µm. ( C ) PC12 cells were transfected with FGFR1(TK-), FGFR1(SP−/NLS)(TK-) or β-gal. After 24 hr, cells were incubated for an additional 24 hours with or without NGF, and subsequently with 0.1 mM 5′-flurouridine (FU) for 25 minutes to label newly synthesized RNA. FU-RNA was detected using anti-BrdU and confocal microscopy. Bars represent average (±SEM) intensity of nuclear 5′-FU-IR in at least 19 EGFP expressing cells. NGF significantly increased FU-IR intensity in PC12 cells. Dominant negative FGFR1 significantly decreased the FU-IR intensity in NGF treated and non-treated cells. One-Way ANOVA, LSD: *p<0.05 difference between – NGF and+NGF within individual plasmid groups; X p<0.05 difference from β-gal -NGF; #p<0.05 difference from β-gal +NGF. Two-Way ANOVA (p = 0.057) suggests a potential interaction between transfected plasmid DNA and NGF. Bar length - 10 µm.

Techniques: Incubation, Quantitative RT-PCR, Transfection, Recombinant, Control, Fluorescence, Plasmid Preparation, Synthesized, Confocal Microscopy, Expressing, Dominant Negative Mutation

( A ) In vivo validation of FGFR1 and Nur binding to an NBRE-like site within intron 1 of the DCX gene. Chromatin was immunoprecipitated with FGFR1, Nur77/Nurr1Ab or IgG in dissected rat brain regions including, the olfactory bulb (OB) which express the dcx gene at high levels , and in the cortex (CX), cerebellum (CB) and ventral midbrain (VM), which express the dcx gene at lower levels. The cyclophilin A gene, which lacks potential Nur-binding NurRE, NBRE or RARE like elements was used as a control. Samples were combined from two rats and the graphs show ΔΔCt means±SEM of triplicate assays. ( B ) PC 12 cells were incubated with or without NGF for 48 hours. ChIP-qPCR was performed with FGFR1 or Nur77/Nurr1 antibodies or control IgG within diverse NGF-activated genes. Similar as in vivo, no binding to the cyclophilinA gene was observed (not shown). Graphs show means of duplicate or triplicate samples from a representative experiment. Similar changes were observed in three separate experiments. ( C,D ) FGFR1 augments Nur77 NurRE- and NBRE-dependent transcription in PC12 cells. Cells were transfected with NurRE-Luc ( C ) or NBRE-Luc ( D ) and either FGFR1(SP−/NLS) or dominant negative FGFR1(TK-) in the presence or absence of Nur77. The amount of transfected DNA was adjusted to 1 µg per well. One day after transfection, PC12 cells were incubated with NGF (50 ng/ml) or without NGF for an additional 24 hours. Results represent the mean ± SEM from 2 experiments with at least three replicates. One-Way ANOVA, LSD: *p<.001 difference between –NGF and +NGF in individual plasmid transfection groups; #p<.001 difference between Nu77 and Nur77+ FGFR1(SP−/NLS) (or FGFR1(TK-) in the absence of NGF; X p<.001 difference between Nu77 and Nur77+ FGFR1(SP−/NLS) (or FGFR1(TK-) in the presence of NGF. Inserts show the proposed mechanisms of FGFR1 and Nur77 co-operation during NGF activation, including dimer binding NuRE and monomer-binding NBRE.

Journal: PLoS ONE

Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

doi: 10.1371/journal.pone.0068931

Figure Lengend Snippet: ( A ) In vivo validation of FGFR1 and Nur binding to an NBRE-like site within intron 1 of the DCX gene. Chromatin was immunoprecipitated with FGFR1, Nur77/Nurr1Ab or IgG in dissected rat brain regions including, the olfactory bulb (OB) which express the dcx gene at high levels , and in the cortex (CX), cerebellum (CB) and ventral midbrain (VM), which express the dcx gene at lower levels. The cyclophilin A gene, which lacks potential Nur-binding NurRE, NBRE or RARE like elements was used as a control. Samples were combined from two rats and the graphs show ΔΔCt means±SEM of triplicate assays. ( B ) PC 12 cells were incubated with or without NGF for 48 hours. ChIP-qPCR was performed with FGFR1 or Nur77/Nurr1 antibodies or control IgG within diverse NGF-activated genes. Similar as in vivo, no binding to the cyclophilinA gene was observed (not shown). Graphs show means of duplicate or triplicate samples from a representative experiment. Similar changes were observed in three separate experiments. ( C,D ) FGFR1 augments Nur77 NurRE- and NBRE-dependent transcription in PC12 cells. Cells were transfected with NurRE-Luc ( C ) or NBRE-Luc ( D ) and either FGFR1(SP−/NLS) or dominant negative FGFR1(TK-) in the presence or absence of Nur77. The amount of transfected DNA was adjusted to 1 µg per well. One day after transfection, PC12 cells were incubated with NGF (50 ng/ml) or without NGF for an additional 24 hours. Results represent the mean ± SEM from 2 experiments with at least three replicates. One-Way ANOVA, LSD: *p<.001 difference between –NGF and +NGF in individual plasmid transfection groups; #p<.001 difference between Nu77 and Nur77+ FGFR1(SP−/NLS) (or FGFR1(TK-) in the absence of NGF; X p<.001 difference between Nu77 and Nur77+ FGFR1(SP−/NLS) (or FGFR1(TK-) in the presence of NGF. Inserts show the proposed mechanisms of FGFR1 and Nur77 co-operation during NGF activation, including dimer binding NuRE and monomer-binding NBRE.

Techniques: In Vivo, Biomarker Discovery, Binding Assay, Immunoprecipitation, Control, Incubation, ChIP-qPCR, Transfection, Dominant Negative Mutation, Plasmid Preparation, Activation Assay

( A ). Cross-talk between NGF signaling and INFS. NGF stimulates the MAP/ERK pathway resulting in increased RSK1 activity , , which is known to promote the release of newly synthesized FGFR1 from pre-Golgi membranes into the cytosol and accumulation in the cell nucleus . RSK1 binding to FGFR1 in the nucleus decreases FGFR1 mobility promoting the FGFR1 nuclear retention accumulation . Increased nuclear FGFR1 correlates with FGFR1 interaction with Nur proteins , and Nurs also decrease nuclear FGFR1 movement . The FGFR1/Nur complexes generated by NGF stimulation activate Nur-responsive sites on diverse genes. In addition, upregulation of fgf-2 mRNA by NGF could also contribute to FGFR1 nuclear trafficking since the nuclear accumulation and retention of FGFR1 is facilitated by its ligand FGF-2 , (not shown on diagram). ( B ) The end-point ssTF of NGF stimulation, in addition to Nurs, include CREB, AP1, and NFKB . The same end-point factors were shown to be activated by nuclear FGFR1 through the CBP/RSK1-dependent feed forward mechanism . This common INFS step may enable coordinated programing of diverse genes necessary for NGF-induced neuronal differentiation.

Journal: PLoS ONE

Article Title: NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

doi: 10.1371/journal.pone.0068931

Figure Lengend Snippet: ( A ). Cross-talk between NGF signaling and INFS. NGF stimulates the MAP/ERK pathway resulting in increased RSK1 activity , , which is known to promote the release of newly synthesized FGFR1 from pre-Golgi membranes into the cytosol and accumulation in the cell nucleus . RSK1 binding to FGFR1 in the nucleus decreases FGFR1 mobility promoting the FGFR1 nuclear retention accumulation . Increased nuclear FGFR1 correlates with FGFR1 interaction with Nur proteins , and Nurs also decrease nuclear FGFR1 movement . The FGFR1/Nur complexes generated by NGF stimulation activate Nur-responsive sites on diverse genes. In addition, upregulation of fgf-2 mRNA by NGF could also contribute to FGFR1 nuclear trafficking since the nuclear accumulation and retention of FGFR1 is facilitated by its ligand FGF-2 , (not shown on diagram). ( B ) The end-point ssTF of NGF stimulation, in addition to Nurs, include CREB, AP1, and NFKB . The same end-point factors were shown to be activated by nuclear FGFR1 through the CBP/RSK1-dependent feed forward mechanism . This common INFS step may enable coordinated programing of diverse genes necessary for NGF-induced neuronal differentiation.

Techniques: Activity Assay, Synthesized, Binding Assay, Generated

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