fgf receptor 1 d8e4  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Potential implications of granzyme B in keloids and hypertrophic scars through extracellular matrix remodeling and latent TGF-β activation

doi: 10.3389/fimmu.2024.1484462

Figure Lengend Snippet: List of primary and secondary antibodies used for immunohistochemistry (IHC), tyramide signal amplification (TSA), co-immunofluorescence (Co-IF) and Western blotting (WB).

Article Snippet: WB , Rabbit anti-human FGF Receptor 1 (D8E4) , , Cell signaling (mAB#9740) , 1/1,000 in TBS-0.1% Tween + 5% milk , HRP-conjugated goat anti-rabbit , 1/10,000 in TBS-0.1% Tween, 10% milk.

Techniques: Immunohistochemistry, Amplification, Western Blot, Blocking Assay

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: Primary antibodies used for immunostaining and Western blot experiments.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Immunostaining, Western Blot, Ubiquitin Proteomics

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 reduces the number of FGFR1 and FGF2 vesicles and their colocalization in U251 and SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1 fused to enhanced green fluorescent protein (FGFR1-EGFP) and treated with cyanine 3-labeled FGF2 (FGF2-Cy3) for 30 min. Whole-cell analysis of confocal images revealed a reduction in FGFR1 (green) and FGF2 (red) vesicles per cell and their reduced colocalization (yellow) in response to SPRY2-OE. N = 18 experiments, mean ± SEM. *** p < 0.001, **** p < 0.0001. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP and treated with FGF2-Cy3 for 30 min. Whole-cell analysis of confocal images revealed an increase in FGFR1 (green) and FGF2 (red) vesicles per cell as well as their enhanced colocalization (yellow) in response to shSPRY2. N = 15 experiments, mean ± SEM. *** p < 0.001, **** p < 0.0001. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark internalized FGFR1 vesicles colocalizing with FGF2. Scale bar = 4 µm.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Control, Transfection, Labeling, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 inhibits colocalization of clathrin with FGFR1 and FGF2 in U251 and SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against clathrin. Whole-cell analysis of confocal images revealed no change in the number of clathrin vesicles per cell (blue) after SPRY2-OE. The colocalization of clathrin (blue) with FGFR1 (green; colocalization with clathrin = turquoise) and FGF2 (red; colocalization with clathrin = magenta) was reduced in response to SPRY2-OE. N = 6 experiments, mean ± SEM. * p < 0.05. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against clathrin. The number of clathrin vesicles per cell (blue) was not altered with shSPRY2. The colocalization of clathrin (blue) with FGFR1 (green; colocalization with clathrin = turquoise) and FGF2 (red; colocalization with clathrin = magenta) was enhanced with shSPRY2. N = 5 experiments, mean ± SEM. ** p < 0.01. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark internalized FGFR1 and FGF2 vesicles colocalizing with clathrin. Scale bar = 4 µm.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Control, Transfection, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 inhibits colocalization of caveolin-1 with FGFR1 and FGF2 in U251 and SF126 cells and reduces caveolin-1 vesicles. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against caveolin-1. Whole-cell analysis of confocal images revealed a reduction in the number of caveolin-1 vesicles per cell (blue) after SPRY2-OE. The colocalization of caveolin-1 (blue) with FGFR1 (green; colocalization with caveolin-1 = turquoise) and FGF2 (red; colocalization with caveolin-1 = magenta) was reduced in response to SPRY2-OE. N = 6 experiments, mean ± SEM. ** p < 0.01, *** p < 0.001. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP, treated with FGF2-Cy3 for 30 min, and immunostained against caveolin-1. The number of caveolin-1 vesicles per cell (blue) was slightly but not significantly enhanced with shSPRY2. The colocalization of caveolin-1 (blue) with FGFR1 (green; colocalization with caveolin-1 = turquoise) and FGF2 (red; colocalization with caveolin-1 = magenta) was enhanced with shSPRY2. N = 5 experiments, mean ± SEM. * p < 0.05. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark internalized FGFR1 and FGF2 vesicles colocalizing with caveolin-1. Scale bar = 4 µm.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Control, Transfection, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 decreases transferrin-647 uptake and colocalization of transferrin with FGFR1 and FGF2 in U251 and SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP and treated with FGF2-Cy3 for 30 min and with transferrin-647 for 15 min. Whole-cell analysis of confocal images revealed a reduction in the uptake of transferrin vesicles per cell (blue) after SPRY2-OE. The colocalization of transferrin (blue) with FGFR1 (green; colocalization with transferrin = turquoise) and FGF2 (red; colocalization with transferrin = magenta) was also reduced in response to SPRY2-OE. N = 6 experiments, mean ± SEM. ** p < 0.01, *** p < 0.001. ( B ) SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP and treated with FGF2-Cy3 for 30 min and with transferrin-647 for 15 min. The uptake of transferrin vesicles per cell (blue) was increased with shSPRY2, and the colocalization of transferrin (blue) with FGFR1 (green; colocalization with transferrin = turquoise) and FGF2 (red; colocalization with transferrin = magenta) was enhanced with shSPRY2. N = 5 experiments, mean ± SEM. * p < 0.05, ** p < 0.01. White, bold arrowheads indicate cell surface localization of FGFR1 and FGF2. Yellow arrowheads mark FGFR1 and FGF2 vesicles colocalizing with transferrin. Scale bar = 4 µm.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Control, Transfection, Cell Analysis

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2 reduces FGFR1 and increases EGFR protein but has no effect on FGFR1 and EGFR mRNA levels. ( A ) Western blot analyses of U251 and SF126 cells overexpressing FGFR1-EGFP and treated with FGF2 for 30 min revealed reduced FGFR1 protein after SPRY2-OE in U251 cells and enhanced FGFR1 protein in SF126 cells with shSPRY2. N = 4 experiments, mean ± SD. * p < 0.05. ( B ) qRT-PCR did not reveal changes in FGFR1 mRNA content in response to SPRY2-OE in U251 cells or shSPRY2 in SF126 cells overexpressing FGFR1-EGFP and treated with FGF2 for 30 min. N = 3 experiments, mean ± SEM. ( C ) Western blot analyses of endogenous FGFR1 in U251 and SF126 cells that were not transfected with FGFR1-EGFP but treated with FGF2 for 30 min also revealed reduced FGFR1 protein after SPRY2-OE in U251 cells and enhanced FGFR1 protein in SF126 cells with shSPRY2. N = 4 experiments, mean ± SD. * p < 0.05. ( D ) Western blot analyses of U251 cells overexpressing EGFR-EGFP and treated with EGF for 30 min revealed increased EGFR protein after SPRY2-OE, whereas SF126 cells with shSPRY2 exhibited reduced EGFR protein. N = 3 experiments, mean ± SD. * p < 0.05. ( E ) qRT-PCR did not reveal changes in EGFR mRNA content in response to SPRY2-OE in U251 cells or shSPRY2 in SF126 cells overexpressing EGFR-EGFP and treated with EGF for 30 min. N = 3 experiments, mean ± SEM.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Western Blot, Quantitative RT-PCR, Transfection

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2-OE increases FGFR1 degradation by enhanced c-CBL-mediated ubiquitination and reduces binding of PLCγ1 to FGFR1 in U251 cells. ( A ) Whole-cell lysates of U251 control cells and U251 cells with SPRY2-OE transfected with FGFR1-EGFP and treated with FGF2 for 10 min. U251 cells with SPRY2-OE revealed reduced FGFR1 protein but no change in ubiquitin, c-CBL, or PLCγ1. ( B ) Anti-FGFR1 immunoprecipitates (IP) revealed reduced FGFR1 protein but enhanced ubiquitination and c-CBL after SPRY2-OE. PLCγ1 was reduced in anti-FGFR1 immunoprecipitates with SPRY2-OE, and the overexpressed but not the endogenous SPRY2 was detected in anti-FGFR1 immunoprecipitates. ( C ) Quantification of anti-FGFR1 immunoprecipitates (IP) confirmed the increase of ubiquitin and c-CBL but the reduction of PLCγ1 in response to SPRY2-OE. N = 3 experiments, mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Ubiquitin Proteomics, Binding Assay, Control, Transfection

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SiRNA-induced knockdown of c-CBL increases FGFR1 protein in U251 cells with SPRY2-OE but the mutant SPRY2 Y55F , which does not bind c-CBL, reduces FGFR1 protein. ( A ) Western blot analyses of U251 cells transfected with scrambled control siRNA (siControl) or siRNA against c-CBL (siCBL) overexpressing FGFR1-EGFP and treated with FGF2 for 30 min revealed reduced c-CBL with siCBL compared to siControl in U251 control cells and in U251 cells with SPRY2-OE. N = 4 experiments, mean ± SD. ** p < 0.01. ( B ) U251 cells transfected with control siRNA (siControl) and siRNA against c-CBL (siCBL) overexpressing FGFR1-EGFP and treated with FGF2 for 30 min revealed increased FGFR1 protein in U251 control cells and in U251 cells with SPRY2-OE in response to siCBL. N = 5 experiments, mean ± SD. * p < 0.05, *** p < 0.001. ( C ) FGFR1 protein was reduced by SPRY2-OE and by overexpression of mutant SPRY2 Y55F in U251 cells overexpressing FGFR1-EGFP and treated with FGF2 for 30 min. N = 3 experiments, mean ± SD. ** p < 0.01.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Knockdown, Mutagenesis, Western Blot, Transfection, Control, Over Expression

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2-OE inhibits FGF2-induced activation of PLCγ1 and ERK in U251 cells, but shSPRY2 only increases FGF2-induced PLCγ1 activation in SF126 cells. ( A ) U251 control cells with low endogenous SPRY2 level and U251 cells with SPRY2-OE transfected with FGFR1-EGFP and treated with FGF2 for 30 and 120 min. Activation of PLCγ1 and ERK was inhibited by SPRY2-OE. N = 4 experiments, mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( B ) Naive SF126 control cells with high endogenous SPRY2 content and SF126 cells with shSPRY2 transfected with FGFR1-EGFP and treated with FGF2 for 30 and 120 min. Activation of PLCγ1 but not of ERK was elevated by shSPRY2. N = 4 experiments, mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Activation Assay, Control, Transfection

Journal: Cells

Article Title: Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells

doi: 10.3390/cells13231967

Figure Lengend Snippet: SPRY2-OE increases cell viability and reduces stemness markers in U251 cells, whereas shSPRY2 enhances cisplatin sensitivity and increases stemness markers in SF126 cells. ( A ) U251 control cells and U251 cells with SPRY2-OE were transfected with FGFR1-EGFP and treated with FGF2 and 15 and 30 µM cisplatin for 24 h. Cell viability of U251 cells was increased by SPRY2-OE in all groups. N = 4 experiments, mean ± SEM. **** p < 0.0001. ( B ) Naive SF126 control cells and SF126 cells with shSPRY2 were transfected with FGFR1-EGFP and treated with FGF2 and 10 and 15 µM cisplatin for 24 h. Cell viability of SF126 cells was not altered by shSPRY2, but cell viability was more strongly reduced by cisplatin treatment in cells with shSPRY2 than in control cells. N = 5 experiments, mean ± SEM. ** p < 0.01, **** p < 0.0001. ( C ) SPRY2-OE reduces SOX2 and CD44 in U251 cells overexpressing FGFR1-EGFP. N = 4 experiments, mean ± SD. ** p < 0.01, *** p < 0.001. ( D ) ShSPRY2 increases SOX2 and CD44 in SF126 cells overexpressing FGFR1-EGFP. N = 4 experiments, mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: FGFR1 (D8E4) XP , Cell signaling , 9740.

Techniques: Control, Transfection