Journal: Molecular Biomedicine
Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes
doi: 10.1186/s43556-025-00398-w
Figure Lengend Snippet: Impaired activation of signaling pathways by FGF1 variants due to reduced affinity for the FGFR1 (IIIc) receptor. a Serum-starved NIH 3T3 cells were stimulated with 10 ng/mL FGF1 variants in the presence of heparin (10 U/mL) for 15 min, and activation of downstream signaling cascades was detected by immunoblotting using the following antibodies: anti-phospho-FGFR (pFGFR), anti-phospho-PLCγ (pPLCγ), anti-phosphoFRS2 (pFRS2), anti-phospho-ERK1/2 (pERK1/2). Anti-ERK1/2, anti-FGFR1, anti-PLCγ and anti-γTubulin antibodies were used to confirm equal loading. Representative results are shown. Densitometric analysis is presented as mean ± SEM, n = 3/4. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001. b BLI analysis of the affinity of FGF1 variants for FGFR1-Fc (IIIc isoform). FGFR1-Fc was immobilized on a Protein A sensor and its interactions (association and dissociation) with selected FGF1 mutants were analyzed in the concentration range of 100–800 nM. Curves obtained by global fitting are marked in red. Representative results are shown (n ≥ 3). The equilibrium dissociation constant (K D ) was calculated from the saturation binding curve
Article Snippet: The following primary antibodies were used: anti-phospho-FGFR (Tyr653/Tyr654) (pFGFR) (#06–1433) from Millipore, anti-tubulin (#T6557) from Sigma-Aldrich, anti-FGFR1 (FGFR1) (#9740), anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase (pERK1/2) (#9101), anti-p44/42 MAP kinase (ERK1/2) (#9102), anti-phospho-FRS2α (Tyr196) (pFRS2) (#3864), anti-vinculin (#13901), anti-phospho-PLCγ1 (Tyr783) (pPLCγ) (#14,008), anti-PLCγ1 (#5690), anti-Glut1 (#73,015) and anti-FGF1 (#3139) from Cell Signaling Technology.
Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Concentration Assay, Binding Assay