rabbit anti phospho fgfr1  (Bioss)

Journal: Cell Death Discovery

Article Title: FGFR inhibition as a new therapeutic strategy to sensitize glioblastoma stem cells to tumor treating fields

doi: 10.1038/s41420-025-02542-5

Figure Lengend Snippet: A Relative RNA expression of FGFR1, FGFR2, FGFR3 and FGFR4 determined by Real-time PCR and expressed as Fold change in GSC compared to FGFR1 expression in GC1. Errors bars show means ± SEM of N = 3 experiments. B Immunoblots of FGFR1, FGFR2, FGFR3 and FGFR4 proteins in GSC. GAPDH was used as loading control. C Relative RNA expression of FGFR1, FGFR2, FGFR3 and FGFR4 determined by Real-time PCR and expressed as Fold change in GC3 and GC4 treated by TTFields (TTFields(+)) compared to control without treatment (TTFields(-)). Errors bas show means ± SEM of N ≥ 3 experiments. D Immunoblots of FGFR1, FGFR2, FGFR3 and FGFR4 proteins in GC3 and GC4 treated with TTFields (TTFields(+)) or untreated (TTFields(-)). GAPDH was used as loading control.

Article Snippet: Membranes were then blocked into 10% milk for 1 h. Primary antibody against Phosphorylated FRS2 (Tyr196) (#3864, Cell Signalling, Danvers, MA, USA), FGFR1 (#9740, Cell Signalling, Danvers, MA, USA), FGFR2 (#A23298, ABclonal, Woburn, MA, USA), FGFR3 (#A0404, ABclonal, Woburn, MA, USA), FGFR4 (#A9197, ABclonal, Woburn, MA, USA) and against GAPDH (#CB1001, Millipore, Molsheim, France) were incubated overnight.

Techniques: RNA Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: A) Distribution of N546K (n = 213) and K656E (n = 121) tumors comparing Brain vs no-brain, or “Others” tumor types. Numbers are specified in . Percentage of cases presenting only the hotspot (single) vs hotspot + other hits in FGFR1 (multiple) are indicated for each group. Both categories (single and multiple) are significantly associated with brain tumor types (Chi-squared test, p value < 0.0001). B) Distribution of N546K and K656E brain tumors comparing low-grade (grade 1-2) vs high-grade (grade 3-4) tumor types. Percentage of cases presenting only the hotspot (single) vs hotspot + other hits in FGFR1 (multiple) are indicated for each group. C) Timeline of acquisition of hotspot (either N546K or K656E) and “secondary” hits. The two possible scenarios with respective examples (familial case and ECCL patients) are indicated.

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques:

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques:

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: Proximal interactome profiling (BioID-MS) of WT and mutant FGFR1 proteins. A) Scheme of the FGFR1 receptor representing functional domains and SNVs investigated in the present study: the hotspots N546K, K656E and the germline variant R661P. B) Stable inducible HEK293 (Flp-In T-REx) cell lines have been generated to express the coding sequence of WT and the 5 mutant FGFR1 fused mutant BirA* enzyme and Flag. C) Western blot of the parental (empty-Flag) and the six Flp-In T-REx HEK293 cell lines used in the present study, showing expression and activation (phosphorylation of Tyr653/654) of FGFR1, after 24h of Tet induction. D) A schematic outline of the BioID-MS screen: in the presence of biotin the BirA* enzyme fused to both active (phosphorylated dimers) and inactive FGFR1 molecules catalyzes the biotinylation of proximal interactors, which are isolated through streptavidin-mediated pull-down and analyzed through MS. E) Number of identified preys (after filtering) as well as BioGRID recalls for each bait are represented. Right: numbers of shared and non-shared interactors of WT FGFR1 between our list and the one published by ( Salokas et al ., 2022 ). F) Dot plot highlighting FGFR1 bona fide interactors and their abundances (AvgSpec) among the different baits. G) Upset plot showing numbers of proximal interactors among different combinations of intersections of the different baits. H) Heatmap of an unsupervised hierarchical clustering analysis using bait abundances (Log2AvgSpec). 17 distinct clusters have been identified. AvgSpec = Average of spectral counts.

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques: Mutagenesis, Functional Assay, Variant Assay, Generated, Sequencing, Western Blot, Expressing, Activation Assay, Phospho-proteomics, Isolation

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: N546K and K656E mutated receptors accumulate at higher levels in HEK293 cells. A) Bar plot of relative amounts (3 technical quantifications of western blots from n = 6 independent experiments) of total Flag-FGFR1 and ratio phospho-Tyr653/654 / Flag-FGFR1 of WT and mutant FGFR1 proteins (as in ) obtained using the six T-REx HEK293 cell lines, representing differences in levels of protein accumulation and intrinsic autophosphorylation, respectively. Fold change (FC) values, obtained normalizing against WT levels, are indicated. Error bars represent standard errors of the mean ± SEM values. Significant comparisons against WT values are indicated (Krustal-Wallis test, p value * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001). B) Dot plot of GO enrichment analysis using the list of genes included in Cluster 11 of the heatmap in (shared interactors among the 4 hotspot-expressing cell lines). The size size and color of dots represent numbers of matched genes and adjusted p-value (p.adjust), respectively. C) Immunofluorescence staining of FGFR1 protein (pink) and α-Tubulin (green) using the T-REx HEK293 FGFR1 cell lines, displaying differences in cellular localization of FGFR1 receptors. Right panels: zoom in (white squares in left panels). Arrows highlight different patterns of FGFR protein in the oncogenic mutants (white arrows indicating accumulation in intracellular compartments), compared with WT and R661P (yellow arrows indicating FGFR1 localization to the plasma membrane). Scale bar: 50 µm. D-E) Western blot assays revealing Flag-FGFR1 expression and tyrosine phosphorylation after 8 hours (D) and 24 hours (E) of Tet-induction. In (E) cells have been treated in parallel with the lysosome inhibitor Bafilomycin A1 (200 nM). F) Time course of Flag-FGFR1 protein synthesis showing differential accumulation rates and patterns among the different mutants and in presence or absence of BafA1. Equal amounts of loaded protein have been assessed through Ponceau staining (Appendix Fig. S3). GO, Gene Ontology.

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques: Western Blot, Mutagenesis, Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Phospho-proteomics

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: R661P reduces K656E and N546K mutant protein stability by rescuing lysosomal degradation. A) Receptor stability experimental design: HEK293 cells expressing WT and mutant FGFR1 are stimulated with human FGF2 (hFGF2, 25 ng/mL) to induce receptor internalization and degradation and, in parallel, with cycloheximide (100 µg/mL) to block protein synthesis. B and D) Western blot analysis of a representative time-course experiment comparing WT FGFR1, K656E (B) and N546K (D) single and double mutants showing total Flag-FGFR1 protein levels at 0, 3 and 6 hours post-treatment with FGF2 and cycloheximide (CHX). C and E) Relative amounts of WT, single and double mutant FGFR1 protein obtained by quantifying western blots from n = 3 independent experiments. Values have been normalized (FC) against each relative 0h reference values. Data is represented by mean ± SEM and significant variations in protein amounts against each specific reference values have been indicated (ANOVA test, p value * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001) in the bar plots.

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques: Mutagenesis, Expressing, Blocking Assay, Western Blot

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: N546K/R661P double mutant interacts with PLC γ with reduced affinity compared to N546K. A) Schematic representation of activated FGFR1 bound to PLCγ (left) and dot plot with relative abundances (AvgSpec) of PLCγ protein for WT and mutant FGFR1 obtained through the BioID-MS screening. B) Predicted 3D structure of FGFR1-PLCγ complex obtained using AF3. The square highlights a zoomed area with the 546 residue in WT (Asn) and mutant (Lys) FGFR1 protein, locating in the interface of FGFR1-PLCγ interaction. The residues Lys656 and Arg661 are also highlighted, to show how residue 546 is the only one close to the interface of interaction and supporting a role for the N546K mutation in enhancing the binding between the two proteins. C) Western blot of total lysates and the co-immunoprecipitation (IP) of total and phosphorylated FGFR1 and PLCγ proteins, using the T-REx HEK293 stable cell lines. AvgSpec = Average of spectral counts.

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques: Mutagenesis, Residue, Binding Assay, Western Blot, Immunoprecipitation, Stable Transfection

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: Defects in self-renewal and changes in phenotype imposed by R661P mutation. A) Schematic representation of the CRISPR design to generate FGFR1 single-and double-mutant clones using the HOG cell line. Genomic deletion in FGFR1 locus in chromosome 8 (chr.8p11.23) identified in these cells has been represented . B) Colony forming assays comparing CRISPR-edited, FGFR1-mutant HOG clones. Both clones for each mutation (N546K, K656E, N546K/R661P, K656E/R661P and R661P) have been assayed. Microscope captures of representative wells of clones #1 for each mutant have been included on the left and quantification of n ≥ 3. experiments have been plotted, normalized against the parental cell line (dashed line). Replicates are represented by black dots. Results of statistical analysis of multiple comparisons (ANOVA test) have been plotted in the heatmap on the right. C) Colony forming assays comparing the two R661P clones with an WT clone and a KO clone. Representative wells of the second R661P clone, WT and KO clones are indicated. Quantification of n ≥ 3. experiments have been plotted, normalized against the parental cell line (dashed line). Results of statistical analysis of multiple comparisons (ANOVA test) have been plotted in the heatmap on the left. D) Matrigel 3D colony forming assays comparing the two R661P clones with the parental cell line. Captures of a representative field displaying colonies formed at experimental endpoint (day 7) are reported on the left. Scale bar: 200 µm. On the right: bar plot of quantified colony areas, normalized against control condition (Parental cell line). Mean ± SEM values (n ≥ 3 independent experiments) and significant differences have been indicated (Krustal-Wallis test, p value * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001).

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques: Mutagenesis, CRISPR, Clone Assay, Microscopy, Control

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: R661P mutant HOG cells show dysregulation of development and neurodevelopment-associated genes, rescued in double mutants. A) Volcano plots representing downregulated (red dots) and upregulated (blue dots) genes differentially expressed genes comparing each R661P clone with the Parental cell line. Numbers of specific and common DE genes are specified in Venn diagrams (right). B) Dot plot of GO enrichment analysis of R661P vs Parental differentially expressed (DE) genes, using common DE genes obtained from both comparisons showed in A) as input list. Significantly enriched (adj. p value ≤ 0.05) have been represented. Size and color of dots represent number of matched genes and adj. p value, respectively. C) Selected GO categories for further analysis are illustrated. RT-qPCR validation of key genes, comparing RNA levels in R66P clones vs Parental HOG cells. Mean ± SEM values from n =2 independent experiments have been plotted and significant differences have been indicated (ANOVA test, p value * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001). E) Dot plots representing Log 2 FC values of RNAseq DE analysis obtained comparing each FGFR1 mutant clone with the Parental cell line. Error bars represent SD among triplicates. Selected genes (n= 64) that have been plotted are unique genes identified in the GO categories listed on the left (selected from GO in B). Left plot: each independent clone has been represented with a different color. Right plot: Single mutant clones (hotspots N546K #1 and #, K656E #1 and #2) have been represented in red; double mutant clones (N546K/R661P #1 and #2, K656E/R661P #1 and #2) have been represented in light orange and R661P #1 and #2 clones have been represented in blue. DE = Differentially expressed

Article Snippet: The primary antibodies used were anti-FLAG (Merck #F3165), anti-FGFR1 (Abcam #ab76464), anti-phospho-FGFR1 Tyr653/654 (Cell Signaling #52928), anti-PLCγ1 (Cell Signaling #5690), anti-phospho-PLCγ1 Tyr783 (Cell Signaling #2821), anti-tubulin (Merck #F6199) and anti-streptavidin-HRP (Thermofisher #21126).

Techniques: Mutagenesis, Quantitative RT-PCR, Biomarker Discovery, Clone Assay