anti–phosphorylated egfr tyr1068 rabbit (d7a5)  (Cell Signaling Technology Inc)

Journal: Oncology Reports

Article Title: Thermal cycling?hyperthermia sensitizes non?small cell lung cancer A549 cells to EGFR tyrosine kinase inhibitor erlotinib

doi: 10.3892/or.2025.8891

Figure Lengend Snippet: Effect of Erl combined with TC-HT on p-EGFR protein expression in A549 cells. Western blotting of p-EGFR protein expression in A549 cells treated with 10 µM Erl, moderate temperature TC-HT, and the combination treatment. The expression levels of p-EGFR were normalized to GAPDH. Each relative expression level was compared with the control and represented as fold relative to the control. Data are shown as mean ± standard deviation (n=3). Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test. **P<0.01, ***P<0.001. Erl, erlotinib; TC-HT, thermal cycling-hyperthermia; p, phosphorylated; t, total; ctrl, control.

Article Snippet: The specific primary antibodies against phosphorylated EGFR (p-EGFR; cat. no. 4407; Cell Signaling Technology, Inc.), poly ADP-ribose polymerase (PARP; cat. no. 9542; Cell Signaling Technology, Inc.), p-JNK (cat. no. 4668; Cell Signaling Technology, Inc.), p-Akt (cat. no. 4060; Cell Signaling Technology, Inc.), total Akt (t-Akt; cat. no. 9272; Cell Signaling Technology, Inc.), MutT homolog 1 (MTH1; cat. no. 43918; Cell Signaling Technology, Inc.), p-p38 (cat. no. GTX133460; GeneTex, Inc.), Cdc2 (cat. no. GTX108120; GeneTex, Inc.) and GAPDH (cat. no. GTX100118; GeneTex, Inc.) were used.

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Cell communication and signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling.

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Fig. 3 Recombinant REG3A stimulates PANC-1 cell growth via EGFR-MAPK pathways. A-D PANC-1 cells were treated with different concentrations of recombinant human REG3A (rhREG3A) for different durations and the cell viability, colony formation, EdU staining, and cell invasion were determined, **p < 0.01, ***p < 0.001 in 1 μg/mL group, #p < 0.05, ##p < 0.01, ###p < 0.001 in 10 μg/mL group, compared to the vehicle-treated group, n = 5. E PANC-1 cells were treated with 10 μg/mL rhREG3A for 24 h, total RNA was isolated and RNA sequencing was performed. F Volcano plot showing the upregulated and downregulated genes in PANC-1 cells treated with 10 μg/mL rhREG3A for 24 h. G Heatmap showing the representative upregulated genes in rhREG3A-treated PANC-1 cells. H GO/KEGG enrichment analysis showing upregulated pathways in rhREG3A-treated PANC-1 cells. I Protein–protein interaction (PPI) analysis was performed using upregulated genes in the epidermal growth factor (EGF) pathway. J Gene set enrichment analysis (GSEA) of the upregulated genes in rhREG3A-treated PANC-1 cells. K Representative GSEA curves in (J)

Article Snippet: After blocking with bovine serum albumin (BSA), the membranes were incubated with anti-REG3A (#ab192224, Abcam, Cambridge, UK), anti-tubulin (#2128, Cell Signaling Technology, Danvers, MA, US), anti-phosphorylated STAT3 (#9131, Cell Signaling Technology), anti-STAT3 (#4904, Cell Signaling Technology), antiphosphorylated tyrosine (#9411, Cell Signaling Technology), anti-phosphorylated EGFR (Y1068, #3777, Cell Signaling Technology), anti-EGFR (#4405, Cell Signaling Technology), anti-phosphorylated ERK (#4370, Cell Signaling Technology), or anti-ERK (#4695, Cell Signaling Technology) antibodies.

Techniques: Recombinant, Staining, Isolation, RNA Sequencing

Journal: Cell communication and signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling.

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Fig. 4 EGFR-MAPK activation is involved in the proliferative effect of REG3A in PDAC cells. A Phosphokinase array analysis of PANC-1 cells serum starved for 24 h, followed by 10 μg/mL rhREG3A or vehicle treatment for 30 min. Dot blots are shown, with altered dots marked by red frames. B Phosphorylation of tyrosine kinase and C phosphorylation of EGFR and ERK detected by western blots of PANC-1 cells serum starved for 24 h, followed by 10 μg/mL rhREG3A treatment for different times. D Dimerization assay performed using the BS3 cross-linker, followed by western blot analysis using EGFR antibody in PANC-1 cells serum starved for 24 h, followed by 10 μg/mL rhREG3A or 100 ng/mL EGF treatment for 30 min. All blots were repeated at least 3 times; tubulin was used as an internal reference; the integrated optical density (IOD) of each band in the independent blot was analyzed (*p < 0.05, ***p < 0.001, compared to 0-time, n = 3). E The PANC-1 cells were treated with vehicle or 10 μg/mL rhREG3A for 1 h, then the cells were lysed for Co-immunoprecipitation (Co-IP). F Double immunofluorescent staining of REG3A (red) and EGFR (green), in vehicle or rhREG3A-simulated PANC-1 cells, DAPI (blue) was used to stain nuclei, white marker indicated the co-localization of REG3A and EGFR (yellow), scale bar = 20 μm. G The in vitro binding activity of rhREG3A to the extracellular domain of EGFR protein by MST assay

Article Snippet: After blocking with bovine serum albumin (BSA), the membranes were incubated with anti-REG3A (#ab192224, Abcam, Cambridge, UK), anti-tubulin (#2128, Cell Signaling Technology, Danvers, MA, US), anti-phosphorylated STAT3 (#9131, Cell Signaling Technology), anti-STAT3 (#4904, Cell Signaling Technology), antiphosphorylated tyrosine (#9411, Cell Signaling Technology), anti-phosphorylated EGFR (Y1068, #3777, Cell Signaling Technology), anti-EGFR (#4405, Cell Signaling Technology), anti-phosphorylated ERK (#4370, Cell Signaling Technology), or anti-ERK (#4695, Cell Signaling Technology) antibodies.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining, Marker, In Vitro, Binding Assay, Activity Assay

Journal: Cell communication and signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling.

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Fig. 6 REG3A binds to EGFR-ECD region, requiring its C-type lectin domain. A Schematic diagram of full-length (FL), C-terminal deletion (△C), N-terminal deletion (△N) constructs of Flag-REG3A; FL, extracellular domain (ECD), intracellular domain (ICD) of His-EGFR; signal peptide (SP), transmembrane (TM), juxtamembrane (JM) regions, kinase domain (KD), C-terminal region (CR). B, C After the CHO cells were co-transfected with different REG3A/EGFR truncations, anti-His antibody immunoprecipitation (IP: His) was performed. Representative immunoblot of EGFR-His and REG3A-Flag in IP and in whole-cell lysate (input) is shown. D The in vitro binding activity of rhREG3A to the ECD or the ICD of EGFR protein by MST assay, n = 3. E The in vitro binding activity of rhREG3A to the ECD of EGFR protein in the presence/absence of 10 ng/mL EGF by MST assay, n = 3. F The PANC-1 cells were starved for 24 h, and treated with 10 ng/mL EGF in the present/absent of 10 μg/mL rhREG3A for 5 min, or different times, the phosphorylation of EGFR and ERK were determined by western blotting. All blots were repeated at least 3 times; tubulin was used as an internal reference; the integrated optical density (IOD) of each band in the independent blot was analyzed (**p < 0.01, ***p < 0.001, as indicated, n = 3)

Article Snippet: After blocking with bovine serum albumin (BSA), the membranes were incubated with anti-REG3A (#ab192224, Abcam, Cambridge, UK), anti-tubulin (#2128, Cell Signaling Technology, Danvers, MA, US), anti-phosphorylated STAT3 (#9131, Cell Signaling Technology), anti-STAT3 (#4904, Cell Signaling Technology), antiphosphorylated tyrosine (#9411, Cell Signaling Technology), anti-phosphorylated EGFR (Y1068, #3777, Cell Signaling Technology), anti-EGFR (#4405, Cell Signaling Technology), anti-phosphorylated ERK (#4370, Cell Signaling Technology), or anti-ERK (#4695, Cell Signaling Technology) antibodies.

Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Activity Assay, Phospho-proteomics

Journal: Cell communication and signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling.

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Fig. 8 Peritumoral acinar cell secreted REG3A activates EGFR-MAPK signal in PDAC. A The HE staining and immunohistochemical staining using REG3A, CK7, and KI67 antibodies in a same PDAC tissue with different magnifications. B Pathway enrichment in patients with high expression of REG3A in CRA001160 dataset. C Immunohistochemical staining of pERK and pEGFR in patients with different expression of REG3A (left) and vector or REG3A transfected PANC1 xenografts in nude mice (right). E Cell communications between different cell clusters in REG3ALow and REG3AHigh patients in CRA001160 dataset. F Graphic abstract. Inflammatory signals-stimulated STAT3 activation contributes to the increased REG3A expression in acinar cells, which then the secreted REG3A stimulates the growth of PDAC cell by activating EGFR signal directly

Article Snippet: After blocking with bovine serum albumin (BSA), the membranes were incubated with anti-REG3A (#ab192224, Abcam, Cambridge, UK), anti-tubulin (#2128, Cell Signaling Technology, Danvers, MA, US), anti-phosphorylated STAT3 (#9131, Cell Signaling Technology), anti-STAT3 (#4904, Cell Signaling Technology), antiphosphorylated tyrosine (#9411, Cell Signaling Technology), anti-phosphorylated EGFR (Y1068, #3777, Cell Signaling Technology), anti-EGFR (#4405, Cell Signaling Technology), anti-phosphorylated ERK (#4370, Cell Signaling Technology), or anti-ERK (#4695, Cell Signaling Technology) antibodies.

Techniques: Staining, Immunohistochemical staining, Expressing, Plasmid Preparation, Transfection, Activation Assay

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: Frontiers in Pharmacology

Article Title: Oxyresveratrol as a novel ferroptosis inducer exhibits anticancer activity against breast cancer via the EGFR/PI3K/AKT/GPX4 signalling axis

doi: 10.3389/fphar.2024.1527286

Figure Lengend Snippet: ORes induces ferroptosis in breast cancer cells via the EGFR/PI3K/AKT/GPX4 signalling axis. (A) The table indicates the top 10 connectivity scores between the input gene signature and the gene signatures of compounds in the LINCS L1000 touchstone dataset. (B) The table displays the top 10 scores between the input gene signature and the gene signatures of compounds in the DGDB dataset. (C) GSEA of HTS 2 results of Osimertinib dimesylate–treated MDA-MB-231 cells. (D) GSEA of HTS 2 results of AZ-5104–treated MDA-MB-231 cells. (E) Volcano plot of the DEGs in EGFR knockdown MDA-MB-231 cells, with red and blue dots indicating upregulated and downregulated genes, respectively. Differential gene screening criteria: |FoldChange|>1.3 and p- value < 0.05. (F) Representative Western blot results. (G) Quantification of western blots. Experiments were performed in triplicate, and data are presented as mean ± SD. *** p < 0.001; ns, no significance.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against GPX4 (Cell Signaling Technology, 59735, 1:1,000), phosphorylated EGFR (ZenBio, R26283, 1:1,000), EGFR (Selleck, A5858, 1:1,000), phosphorylated PI3K (ZenBio, 310164, 1:1,000), PI3K (ZenBio, 200900, 1:1,000), phosphorylated AKT (Cell Signaling Technology, 13038, 1:1,000), and AKT (Cell Signaling Technology, 4691, 1:1,000).

Techniques: Knockdown, Western Blot