phosphorylated egfr  (Cell Signaling Technology Inc)

Journal: Translational Oncology

Article Title: Novel NOTCH2-NTRK1 fusion confers osimertinib resistance in EGFR-mutant non-small cell lung cancer by interacting with EGFR

doi: 10.1016/j.tranon.2025.102577

Figure Lengend Snippet: NOTCH2-NTRK1 interacts with EGFR protein. (A) Volcano plot of differentially expressed phosphorylated peptides. (blue dots: FC < 0.5 and P value < 0.05; red dots: FC> 2 and P value < 0.05). No significant differences were represented by gray dots. (B) Domain enrichment analysis of proteins with differentially expressed peptides. (C) Phosphorylated sites of protein tyrosine kinases. (D) The expression levels of p-EGFR T669 were assessed via WB analysis in BEAS-2B, H1650, and H1975 cell lines. (E) Co-localization between NOTCH2-NTRK1 with EGFR by immunofluorescence staining. The HA (green) or the EGFR (red) antibody was used to detect NOTCH2-NTRK1 and EGFR protein, respectively. DAPI (blue) was used for nuclei staining. Images are shown at 200 × magnification. (F) Co-IP between NOTCH2-NTRK1 with EGFR in BEAS-2B, H1650, and H1975 cell lines expressing NOTCH2-NTRK1. (IP: immunoprecipitation, IB: immunoblotting).

Article Snippet: Antibodies for total AKT (#4691), phosphorylated AKT (Ser473) (#4060), total ERK (#4695), phosphorylated ERK (Thr202/Tyr204) (#4370), HA (C29F4) (#3724), EGFR (D38B1) (#4267), phosphorylated EGFR (Thr669) (D2F1) (#8808), Met (D1C2) (#8198) and phosphorylated TRKA (Tyr490) (#4619) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot

Journal: Translational Oncology

Article Title: Novel NOTCH2-NTRK1 fusion confers osimertinib resistance in EGFR-mutant non-small cell lung cancer by interacting with EGFR

doi: 10.1016/j.tranon.2025.102577

Figure Lengend Snippet: The mutant NOTCH2-NTRK1 protein, ΔY490, reverses NSCLC cells resistance to osimertinib. (A) The 3D binding model of EGFR with the NOTCH2-NTRK1 fusion protein. EGFR was colored in marine, residues surrounding the binding pockets were shown as green sticks, and those of NTRK1 were shown as red sticks. The hydrogen bonds were depicted as green dashed lines. (B) Amino acid sequence of the NOTCH2-NTRK1 fusion protein. The predicted seven amino acid residues interacting with EGFR protein were colored in red. Tyrosine at position 490 in the intact NTRK1 protein (corresponding to position 329 in the NOTCH2-NTRK1 fusion protein) is marked in green. The truncated region (from Asn304 to Glu409) is underlined. (C) The co-localization between ΔY490 or Y490A variant with EGFR assessed by immunofluorescence staining. The HA (green) antibody was used to detect ΔY490 or Y490A variant. The EGFR (red) antibody was used to detect EGFR protein. DAPI (blue) was used to stain nuclei. Images were shown at 200 × magnification. (D) Co-IP between ΔY490 and Y490A variant with EGFR in BEAS-2B and H1975 cell lines. (IP: immunoprecipitation, IB: immunoblotting) (E) Dose-response curves of osimertinib for H1975-NOTCH2-NTRK1 variant cell lines. (F) Adhesion-dependent colony formation assay following 100 nM osimertinib treatment. ** P < 0.01, compared with the NOTCH2-NTRK1 group; ns , not significant, P ≥ 0.05; Data were presented as means ± SD from three biological replicates. (G) WB analysis of p-AKT, p-ERK1/2, p-EGFR (T669), and p-TRKA (Y490) protein levels assessed in H1975-NOTCH2-NTRK1 variant groups at 0, 0.5, 1, and 4 h after 100 nM osimertinib treatment. Grayscale values of the protein bands were measured using ImageJ software. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Data were presented as means ± SD from two biological replicates.

Article Snippet: Antibodies for total AKT (#4691), phosphorylated AKT (Ser473) (#4060), total ERK (#4695), phosphorylated ERK (Thr202/Tyr204) (#4370), HA (C29F4) (#3724), EGFR (D38B1) (#4267), phosphorylated EGFR (Thr669) (D2F1) (#8808), Met (D1C2) (#8198) and phosphorylated TRKA (Tyr490) (#4619) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Binding Assay, Sequencing, Variant Assay, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Colony Assay, Software

Journal: Frontiers in Pharmacology

Article Title: Cepharanthine may inhibit the proliferation of prostate cells by blocking the EGFR/PI3K/AKT signaling pathway: comprehensive network analysis, molecular docking, and experimental evaluation

doi: 10.3389/fphar.2025.1654757

Figure Lengend Snippet: Western blot confirmed that CEP can inhibit the proliferation of WPMY-1 by inhibiting the EGFR/PI3K/AKT signaling pathway. (A) Western blot analysis showed that CEP could significantly inhibit the expression of FN1 and also had a certain inhibitory effect on the total protein expression of AKT and EGFR, but there was no statistical difference; (B) Compared with the DMSO group, 2.5 μM CEP treatment for 48 h significantly reduced the expression of p-AKT and p-EGFR, and 5 μM CEP further aggravated the inhibitory effect; (C) Quantitative analysis of AKT, p-AKT, EGFR, p-EGFR and FN1 by Western blot; (D) phosphorylation level analysis simultaneously showed that the ratios of p-AKT/AKT and p-EGFR/EGFR in the CEP group were significantly decreased. Data are presented as mean ± SD, and were analyzed with One-way ANOVA with Tukey’s post-hoc test. ****p < 0.0001. NS: Non-significant; Cep: Cepharanthine.

Article Snippet: Antibodies against AKT (No. 75692), phosphorylated AKT (Ser473) (No. 4060), EGFR (No. 4267), phosphorylated EGFR (Tyr1068) (No. 3777), FN1 (No. A0056-3), and GAPDH (No. 2118) were purchased from Cell Signaling Technology, Inc. (Boston, USA).

Techniques: Western Blot, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Identification of internalizing ScFvs for EGFR inhibition and apoptosis induction in cholangiocarcinoma cells

doi: 10.1038/s41598-025-24324-w

Figure Lengend Snippet: Inhibition of EGFR-TK enzyme activity and EGFR phosphorylation by scFv antibodies and erlotinib. ( A – C ) Dose-dependent inhibition of EGFR-TK activity by G8, H2, and erlotinib, and the half-maximal inhibitory concentrations (IC 50 ) are shown. ( D ) Western blot analysis of total EGFR and phosphorylated EGFR (pEGFR) in HuCCA-1 cells treated with E1, G8, H2 or erlotinib. β-actin was used as a loading control. Band intensities were quantified by densitometry, and the ratio of pEGFR to total EGFR is presented as mean ± SEM. Data are representative of three independent experiments. * indicates statistically significant differences compared to control ( P < 0.05). The original Western blot images are shown in Supplementary Fig. .

Article Snippet: Membranes were probed with primary antibodies against phosphorylated EGFR (Y1173) (p-EGFR, 1:500) and total EGFR (1:500) (Santa Cruz Biotechnology, CA, USA). β-actin (1:1000, Santa Cruz Biotechnology) was used as a loading control.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Western Blot, Control