pfgfr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pfgfr
    Pfgfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pfgfr  (Cell Signaling Technology Inc)


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    amino acids  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc amino acids
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    anti fgfr tyr653 654  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti fgfr tyr653 654
    Anti Fgfr Tyr653 654, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    y653  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc y653
    A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation <t>Y653/654</t> (3471, p-FGFR2) and Actin.
    Y653, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival"

    Article Title: FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098515

    A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation Y653/654 (3471, p-FGFR2) and Actin.
    Figure Legend Snippet: A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation Y653/654 (3471, p-FGFR2) and Actin.

    Techniques Used: Microscopy, Labeling, Protein Concentration, SDS Page, Western Blot, Activation Assay

    A. PD173074 and MK2461 inhibit FGFR2 phosphorylation. Cells were treated for 1 hour with a titration of PD173074 as described in Materials and methods. Lysates were prepared and 50 ug protein was subjected to SDS-PAGE and western blotting with phospho Y653/654 FGFR and MAB6841 total FGFR2 antibody. B. PD173074 and MK2461 inhibit NCI-H716 cell growth. Cell lines were plated at 4000 cells/well and incubated overnight. NCI-H716 cells were treated with a titration of PD173074 as described in Materials and Methods. Cell growth was measured with Vialight reagent, and growth was presented relative to untreated cells. C. PD173074 and MK2461 selectively inhibit growth of NCI-H716 cells. Colon cancer cell lines listed were plated at 4000 cells/well and 24 hours later were treated with a titration of compounds. 4 days later cell growth was measured with vialight and IC50s were calculated from graph pad prism. D. FGFR2 shRNA decreases FGFR2 protein in NCI-H716 cells. FGFR2 shRNA was prepared and NCI-H716 cells were infected as described in methods. Left, FGFR2 expression was analyzed with phospho Y653/654 and total protein with MAB6841. E. Growth was analyzed 5 days post infection with Vialight reagent.
    Figure Legend Snippet: A. PD173074 and MK2461 inhibit FGFR2 phosphorylation. Cells were treated for 1 hour with a titration of PD173074 as described in Materials and methods. Lysates were prepared and 50 ug protein was subjected to SDS-PAGE and western blotting with phospho Y653/654 FGFR and MAB6841 total FGFR2 antibody. B. PD173074 and MK2461 inhibit NCI-H716 cell growth. Cell lines were plated at 4000 cells/well and incubated overnight. NCI-H716 cells were treated with a titration of PD173074 as described in Materials and Methods. Cell growth was measured with Vialight reagent, and growth was presented relative to untreated cells. C. PD173074 and MK2461 selectively inhibit growth of NCI-H716 cells. Colon cancer cell lines listed were plated at 4000 cells/well and 24 hours later were treated with a titration of compounds. 4 days later cell growth was measured with vialight and IC50s were calculated from graph pad prism. D. FGFR2 shRNA decreases FGFR2 protein in NCI-H716 cells. FGFR2 shRNA was prepared and NCI-H716 cells were infected as described in methods. Left, FGFR2 expression was analyzed with phospho Y653/654 and total protein with MAB6841. E. Growth was analyzed 5 days post infection with Vialight reagent.

    Techniques Used: Titration, SDS Page, Western Blot, Incubation, shRNA, Infection, Expressing

    anti phosphorylated fgfr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phosphorylated fgfr
    A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. <t>Tyrosine</t> <t>phosphorylated</t> (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of <t>FGFR</t> phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.
    Anti Phosphorylated Fgfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity"

    Article Title: Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110345

    A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. Tyrosine phosphorylated (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of FGFR phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.
    Figure Legend Snippet: A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. Tyrosine phosphorylated (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of FGFR phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.

    Techniques Used: shRNA, Software, Western Blot, Expressing, Blocking Assay

    phospho fgfr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho fgfr
    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and <t>FGFR-dependent</t> signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH <t>and</t> <t>β-tubulin</t> were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.
    Phospho Fgfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Inhibition of autophagy potentiates the cytotoxicity of the irreversible FGFR1-4 inhibitor FIIN-2 on lung adenocarcinoma"

    Article Title: Inhibition of autophagy potentiates the cytotoxicity of the irreversible FGFR1-4 inhibitor FIIN-2 on lung adenocarcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-05201-0

    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH and β-tubulin were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.
    Figure Legend Snippet: a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH and β-tubulin were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

    Techniques Used: Quantitative RT-PCR, Immunohistochemistry, Expressing, Staining, Western Blot

    phospho fgfr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho fgfr
    LHX2 transcriptionally activates FGF1 expression. Tumour cells produce FGF1, which binds to <t>FGFR,</t> and the subsequent downstream signalling occurs through the intracellular receptor substrates FGFR substrate 2 (FRS2), resulting in the phosphorylation and activation of <t>MAPK/ERK,</t> <t>JAK2/STAT3,</t> and PI3K/AKT signalling pathways. (i) These pro-survival pathways are responsible for the promotion of tumour proliferation and growth. (ii) Phosphorylated AKT stimulates the Ser9-GSK3β/β-catenin signalling, leading to the EMT of NPC via the β-catenin targeted ZEB1 and TWIST1 genes and promotes tumour cell migration and invasion. (iii) FGF/FGFR trapping by AZD4547 could block the LHX2/FGF1-induced promotion on tumour growth and metastasis.
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    1) Product Images from "LHX2 facilitates the progression of nasopharyngeal carcinoma via activation of the FGF1/FGFR axis"

    Article Title: LHX2 facilitates the progression of nasopharyngeal carcinoma via activation of the FGF1/FGFR axis

    Journal: British Journal of Cancer

    doi: 10.1038/s41416-022-01902-7

    LHX2 transcriptionally activates FGF1 expression. Tumour cells produce FGF1, which binds to FGFR, and the subsequent downstream signalling occurs through the intracellular receptor substrates FGFR substrate 2 (FRS2), resulting in the phosphorylation and activation of MAPK/ERK, JAK2/STAT3, and PI3K/AKT signalling pathways. (i) These pro-survival pathways are responsible for the promotion of tumour proliferation and growth. (ii) Phosphorylated AKT stimulates the Ser9-GSK3β/β-catenin signalling, leading to the EMT of NPC via the β-catenin targeted ZEB1 and TWIST1 genes and promotes tumour cell migration and invasion. (iii) FGF/FGFR trapping by AZD4547 could block the LHX2/FGF1-induced promotion on tumour growth and metastasis.
    Figure Legend Snippet: LHX2 transcriptionally activates FGF1 expression. Tumour cells produce FGF1, which binds to FGFR, and the subsequent downstream signalling occurs through the intracellular receptor substrates FGFR substrate 2 (FRS2), resulting in the phosphorylation and activation of MAPK/ERK, JAK2/STAT3, and PI3K/AKT signalling pathways. (i) These pro-survival pathways are responsible for the promotion of tumour proliferation and growth. (ii) Phosphorylated AKT stimulates the Ser9-GSK3β/β-catenin signalling, leading to the EMT of NPC via the β-catenin targeted ZEB1 and TWIST1 genes and promotes tumour cell migration and invasion. (iii) FGF/FGFR trapping by AZD4547 could block the LHX2/FGF1-induced promotion on tumour growth and metastasis.

    Techniques Used: Expressing, Activation Assay, Migration, Blocking Assay

    pfgfr y653 654  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pfgfr y653 654
    Pfgfr Y653 654, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    psmad2 s465  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc psmad2 s465
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    p fgfr y653 654  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p fgfr y653 654
    P Fgfr Y653 654, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation <t>Y653/654</t> (3471, p-FGFR2) and Actin.
    Y653, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phosphorylated fgfr
    A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. <t>Tyrosine</t> <t>phosphorylated</t> (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of <t>FGFR</t> phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.
    Anti Phosphorylated Fgfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and <t>FGFR-dependent</t> signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH <t>and</t> <t>β-tubulin</t> were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.
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    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and <t>FGFR-dependent</t> signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH <t>and</t> <t>β-tubulin</t> were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.
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    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and <t>FGFR-dependent</t> signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH <t>and</t> <t>β-tubulin</t> were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.
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    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and <t>FGFR-dependent</t> signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH <t>and</t> <t>β-tubulin</t> were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.
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    Image Search Results


    A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation Y653/654 (3471, p-FGFR2) and Actin.

    Journal: PLoS ONE

    Article Title: FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

    doi: 10.1371/journal.pone.0098515

    Figure Lengend Snippet: A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation Y653/654 (3471, p-FGFR2) and Actin.

    Article Snippet: Antibodies against FGFR2 included N-terminus MAB6841 (R&D Systems, Minneapolis, MN) and H80 (Santa Cruz), C-terminus C-20 (Santa Cruz) and Y653/654 specific 3471 ((Cell Signaling Technology, (CST), Danvers MA.)).

    Techniques: Microscopy, Labeling, Protein Concentration, SDS Page, Western Blot, Activation Assay

    A. PD173074 and MK2461 inhibit FGFR2 phosphorylation. Cells were treated for 1 hour with a titration of PD173074 as described in Materials and methods. Lysates were prepared and 50 ug protein was subjected to SDS-PAGE and western blotting with phospho Y653/654 FGFR and MAB6841 total FGFR2 antibody. B. PD173074 and MK2461 inhibit NCI-H716 cell growth. Cell lines were plated at 4000 cells/well and incubated overnight. NCI-H716 cells were treated with a titration of PD173074 as described in Materials and Methods. Cell growth was measured with Vialight reagent, and growth was presented relative to untreated cells. C. PD173074 and MK2461 selectively inhibit growth of NCI-H716 cells. Colon cancer cell lines listed were plated at 4000 cells/well and 24 hours later were treated with a titration of compounds. 4 days later cell growth was measured with vialight and IC50s were calculated from graph pad prism. D. FGFR2 shRNA decreases FGFR2 protein in NCI-H716 cells. FGFR2 shRNA was prepared and NCI-H716 cells were infected as described in methods. Left, FGFR2 expression was analyzed with phospho Y653/654 and total protein with MAB6841. E. Growth was analyzed 5 days post infection with Vialight reagent.

    Journal: PLoS ONE

    Article Title: FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

    doi: 10.1371/journal.pone.0098515

    Figure Lengend Snippet: A. PD173074 and MK2461 inhibit FGFR2 phosphorylation. Cells were treated for 1 hour with a titration of PD173074 as described in Materials and methods. Lysates were prepared and 50 ug protein was subjected to SDS-PAGE and western blotting with phospho Y653/654 FGFR and MAB6841 total FGFR2 antibody. B. PD173074 and MK2461 inhibit NCI-H716 cell growth. Cell lines were plated at 4000 cells/well and incubated overnight. NCI-H716 cells were treated with a titration of PD173074 as described in Materials and Methods. Cell growth was measured with Vialight reagent, and growth was presented relative to untreated cells. C. PD173074 and MK2461 selectively inhibit growth of NCI-H716 cells. Colon cancer cell lines listed were plated at 4000 cells/well and 24 hours later were treated with a titration of compounds. 4 days later cell growth was measured with vialight and IC50s were calculated from graph pad prism. D. FGFR2 shRNA decreases FGFR2 protein in NCI-H716 cells. FGFR2 shRNA was prepared and NCI-H716 cells were infected as described in methods. Left, FGFR2 expression was analyzed with phospho Y653/654 and total protein with MAB6841. E. Growth was analyzed 5 days post infection with Vialight reagent.

    Article Snippet: Antibodies against FGFR2 included N-terminus MAB6841 (R&D Systems, Minneapolis, MN) and H80 (Santa Cruz), C-terminus C-20 (Santa Cruz) and Y653/654 specific 3471 ((Cell Signaling Technology, (CST), Danvers MA.)).

    Techniques: Titration, SDS Page, Western Blot, Incubation, shRNA, Infection, Expressing

    A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. Tyrosine phosphorylated (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of FGFR phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.

    Journal: PLoS ONE

    Article Title: Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity

    doi: 10.1371/journal.pone.0110345

    Figure Lengend Snippet: A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. Tyrosine phosphorylated (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of FGFR phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.

    Article Snippet: Mouse monoclonal antibody anti-phosphotyrosine (P-Tyr-100), anti-β-actin (8H10D10), anti-Akt, anti-phosphorylated Akt Ser473 (D9E), anti-phosphorylated mitogen-activated protein kinase antibody, anti-phosphorylated-p44/42 MAPK (Erk1/2), anti-phosphorylated EGFR (Tyr1173), anti-Met, anti-phosphorylated Met (Tyr1234/1235), anti-phosphorylated FGFR (Tyr653/654), anti-phosphorylated HERB3/ErbB3 (Tyr1289), anti-phosphorylated-FRS2-α (Tyr196), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: shRNA, Software, Western Blot, Expressing, Blocking Assay

    a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH and β-tubulin were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

    Journal: Cell Death & Disease

    Article Title: Inhibition of autophagy potentiates the cytotoxicity of the irreversible FGFR1-4 inhibitor FIIN-2 on lung adenocarcinoma

    doi: 10.1038/s41419-022-05201-0

    Figure Lengend Snippet: a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH and β-tubulin were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

    Article Snippet: The primary antibodies used in this study were phospho-FGFR (p-FGFR, #3471), p-FRS2 α(#3861), p-ERK1/2 (#4370), β-actin (#3700), and GAPDH (#5174 S), and they were purchased from Cell Signalling Technologies (Danvers, MA, USA). β-tubulin (#BSM-33034) was purchased from Bioss Technology Co., Ltd. (Beijing, China).

    Techniques: Quantitative RT-PCR, Immunohistochemistry, Expressing, Staining, Western Blot