phosphorylated egfr egfr p  (Cell Signaling Technology Inc)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

doi: 10.1016/j.biopha.2024.117605

Figure Lengend Snippet: C12 inhibits the shedding of EGFR ligands and other ADAM17 substrates on the cell surface. Sandwich ELISA was employed to quantitate the shedding or cleavage of EGFR ligands ( A ) and other ADAM17 substrates ( B ). For Notch, we measured the release of the Notch intracellular domain or NICD1 in the cell lysates. Percent inhibition of shedding by the antagonists was calculated by measuring the decrease in shedding observed in comparison to the untreated wells. The data represent mean of quadruplicate determinations and the bars show the effect of administration of antagonists on the different cancer cells (as indicated above the bars) relative to the control (untreated) mean ± SEM.

Article Snippet: We measured the levels of total EGFR and phosphorylated EGFR (EGFR-P), with or without C12 treatment, using sandwich ELISA kits (PathScan Cell signaling technologies) [ ].

Techniques: Sandwich ELISA, Inhibition, Comparison, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

doi: 10.1016/j.biopha.2024.117605

Figure Lengend Snippet: C12 inhibits EGFR/erbB phosphorylation in cancer cells in vitro. Sandwich ELISA was used to measure the levels of total and phosphorylated EGFR (EGFR-P) in MDA-MB-231, HCC827 and SKOV-3 cells upon treatment with 10 μg/ml C12. The data represent mean of triplicate experiments, and the bar plots show the effect of treatment with C12 relative to untreated control, mean ± SEM. Comparison of EGFR levels between treated and untreated groups was performed using independent t test. Total EGFR levels did not significantly differ between the two groups in MDA-MB-231, p=0.85; SKOV-3, p=0.116. However, in HCC827, the small but statistically significant difference could be due to a decrease in the total number of viable cells. On the other hand, the mAb-treated group showed significant decrease of the EGFR-P levels in all cell types, as compared to the untreated control p<0.001, p=0.005.

Article Snippet: We measured the levels of total EGFR and phosphorylated EGFR (EGFR-P), with or without C12 treatment, using sandwich ELISA kits (PathScan Cell signaling technologies) [ ].

Techniques: Phospho-proteomics, In Vitro, Sandwich ELISA, Control, Comparison

Journal: Scientific reports

Article Title: Deoxybouvardin targets EGFR, MET, and AKT signaling to suppress non-small cell lung cancer cells.

doi: 10.1038/s41598-024-70823-7

Figure Lengend Snippet: Fig. 3. Inhibition of protein kinases by DB. (A) In vitro ADP-Glo kinase activity assay was performed in the presence of DB, GEF, SAV, or AZD5363 for kinases EGFR, MET, AKT1, and AKT2. Data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the controls. (B) AutoDock molecular modeling of binding of DB to the kinases. For each kinase, an overview of the surface and cartoon depiction are shown; DB is shown in spheres. Zoomed in: DB in pink sticks and surrounding amino acids in purple sticks.

Article Snippet: Primary antibodies detecting phosphorylated (p)-EGFR (Thr1068), p-MET (Tyr1234/1235), MET, p-AKT (Ser473), and poly ADP-ribose polymerase (PARP) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, In Vitro, Kinase Assay, Binding Assay

Journal: Scientific reports

Article Title: Deoxybouvardin targets EGFR, MET, and AKT signaling to suppress non-small cell lung cancer cells.

doi: 10.1038/s41598-024-70823-7

Figure Lengend Snippet: Fig. 4. Suppression of protein phosphorylation by DB. NSCLC cells HCC827 and HCC827GR treated with DB (0, 2, 4, and 8 nM) for 48 h were harvested and subjected to western blot analysis with antibodies against p-EGFR (Tyr1068), EGFR, p-MET (Tyr1234/1235), MET, p-AKT (Ser473), AKT, and β-actin. (A) Western blot analysis. (B) Relative intensity of p-EGFR/EGFR relative to the level of β-actin. (C) Relative intensity of p-MET/ MET. (D) Relative intensity of p-AKT/AKT. (E, F) HCC827 and HCC827GR cells were treated with or without GEF (1 μM) and the level of kinases EGFR, MET, and AKT was determined by western blotting to examine the relative intensity of phosphoproteins. Data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the controls.

Article Snippet: Primary antibodies detecting phosphorylated (p)-EGFR (Thr1068), p-MET (Tyr1234/1235), MET, p-AKT (Ser473), and poly ADP-ribose polymerase (PARP) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: Scientific reports

Article Title: Deoxybouvardin targets EGFR, MET, and AKT signaling to suppress non-small cell lung cancer cells.

doi: 10.1038/s41598-024-70823-7

Figure Lengend Snippet: Fig. 6. Induction of ROS generation by DB. NSCLC cells HCC827 and HCC827GR treated with DB (0, 2, 4, and 8 nM) for 48 h were analyzed using flow cytometry using a MuseTM Oxidative Stress Kit. (A) Flow cytometry plots and histogram of cellular ROS. ***p < 0.001 compared to the controls. (B and C) NSCLC cells were pretreated with NAC (4 mM) or vehicle for 3 h and then incubated with DB for 48 h. (B) Histogram of cell viability measured by the MTT assay. ***p < 0.001 compared to the controls; ###p < 0.001 compared to DB treatment. (C) Immunoblot analysis of phosphorylated forms of EGFR, MET, AKT, whole caspase-3, and PARP. β-actin was used as the control.

Article Snippet: Primary antibodies detecting phosphorylated (p)-EGFR (Thr1068), p-MET (Tyr1234/1235), MET, p-AKT (Ser473), and poly ADP-ribose polymerase (PARP) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Flow Cytometry, Incubation, MTT Assay, Western Blot, Control

Journal: Scientific reports

Article Title: Deoxybouvardin targets EGFR, MET, and AKT signaling to suppress non-small cell lung cancer cells.

doi: 10.1038/s41598-024-70823-7

Figure Lengend Snippet: Fig. 9. Overview of DB-induced apoptosis in NSCLC cells. Inhibition of EGFR, MET, and AKT kinases by DB triggers cell cycle arrest, ROS production, cytochrome c release, and caspase activation, ultimately leading to the apoptosis induction in NSCLC cells.

Article Snippet: Primary antibodies detecting phosphorylated (p)-EGFR (Thr1068), p-MET (Tyr1234/1235), MET, p-AKT (Ser473), and poly ADP-ribose polymerase (PARP) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activation Assay