Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a, Top: Scheme of metabolomic analysis using extracts from nerve segments. Bottom: Concentrations of key energy metabolism intermediates in control and axotomized nerve segments from C57Bl/6J mice (F6P/G6P: fructose-6-phosphate/glucose-6-phosphate. FBP: fructose-1,6-bisphosphate. GI-OH3P: Glyceraldehyde-3-phosphate. 2PG/3PG: 2-phosphoglycerate/3-phosphoglycerate. LACT: lactate) (Error bars represent s.e.m. n=5 mice per condition and metabolite). b, Top: Scheme of extracellular flux analysis of SCs purified from C57Bl/6J mouse nerves. Bottom: Box and whiskers plot (maximum, 25th percentile, median, 75th percentile, minimum) shows glycolytic activity parameters as assessed by extracellular acidification rate (ECAR) measurements in control mouse SCs and cells with Nrg1-induced ErbB2 activation (n=9 well preparations per condition, *P=0.0080, **P=0.0464, ***P<0.0001). c, Western blot analysis (cropped blot images) of control- and Nrg1-treated C57Bl/6J mouse SCs probed with the indicated antibodies. (n=6 independent pair preparations (2 separate dishes) for PFKFB3, and n=3 independent pair preparations (2 separate dishes) for LDHA quantification). d, Intracellular and extracellular (supernatant) lactate concentrations from control and Nrg1-treated mouse SC preparations normalized to cell number and cellular protein. Note decreased intracellular and increased extracellular lactate levels in SCs treated with Nrg1 for 24h, indicating greatly increased lactate extrusion (Error bars represent s.e.m. n=3 well preparations from 3 independent experiments per condition).

Article Snippet: SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures.

Techniques: Control, Purification, Activity Assay, Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of EGFR downstream signaling in wild type cells and clones upon treatment with EGF. ( A ) Western blots were performed on whole cell lysates isolated from cells serum starved for 1 h and then incubated with recombinant EGF for 30 min in serum free media with 1% BSA. Control cells were incubated in serum free media for 30 min. Experiments were performed independently from at least 3 different passages of cells and representative blots are shown. Antibodies used in this work came from antibody panel ab283852 (Abcam, Cambridge, United Kingdom). ( B ) Quantification of Western blot results was performed using Li-cor quantification software. The results were normalized to protein expression in wild type cells not treated with EGF set at 100%. Data are presented as the mean ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Clone Assay, Western Blot, Isolation, Incubation, Recombinant, Control, Software, Expressing

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of cell cycle distribution and expression of cell cycle regulator proteins. ( A ) Representative cell cycle profiles of wild type ME180 cells and mutant clones based on DAPI staining and incorporation of EdU. The usual horseshoe pattern cell distribution allowed separation of cell populations into G1, S and G2/M stage of the cell cycle. ( B ) Histogram showing cell cycle distribution in wild type cells and mutant clones based on flow cytometry data done on 5 independent experiments using cells from 5 different cell passages. The data are shown as means ± SD from five independent experiments including cells from three different cell cycle passages. ( C ) Western blots performed on whole cell lysates from wild type cells and mutant clones growing in cell culture or additionally treated with recombinant EGF for 30 min. Experiments were performed independently using at least three different cell passages and representative blots are shown. ( D ) Quantification of Western blot results was performed using Li-Cor quantification software and the results are presented values relative to protein expression in untreated wild type cells set as 100%. Data are presented as means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Expressing, Mutagenesis, Clone Assay, Staining, Flow Cytometry, Western Blot, Cell Culture, Recombinant, Software

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 2. Induction of HBEGF ectoshedding by mechanical strain and ET-1. Cardiac myocytes were subjected to a static or strain environment for 0–30 min (panel A) or ET-1 (107 M) for 5 min (panel B), after which medium was collected and concentrated. HBEGF was detected by immunoprecipitation and immunoblotting. Data are ex- pressed as means S.E., n 3. *, p 0.05; **, p 0.01 versus static control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 3. EGFR ligands activate the BNP promoter. Cardiac myo- cytes were co-transfected with 0.5 g of 1595 hBNP-luciferase and 0.05 g of actin -galactosidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and then treated with 0–10 ng/ml EGF (panel A, n 4) or HBEGF (panel B, n 4) for 24 h. Cells were processed and assayed for luciferase activity. Data are expressed as means S.E. *, p 0.05 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 5. Induction of cardiac myo- cyte hypertrophy by HBEGF. After 24 h of culture, cells were treated with HBEGF (1 or 10 ng/ml) or ET-1 (107 M) for 48 h. Panel A, HBEGF stimulates pro- tein synthesis in cultured neonatal rat ventricular myocytes. Following treat- ment, cells were pulsed with [3H]leucine for 4 h, and incorporation of radioactivity into acid-insoluble protein was measured (n 4). Panels B and C, HBEGF in- creases surface area (panel B) and sarco- meric organization (panel C) of cultured neonatal rat ventricular myocytes. Fol- lowing treatment, cells were fixed with 3.7% paraformaldehyde, stained with monoclonal antibody against rat sarcom- eric -actinin, and viewed using fluores- cence microscopy. Panel B, cell surface areas of individual cells were quantified as described under “Experimental Proce- dures” (n 20). Data are expressed as the means S.E. *, p 0.05; **, p 0.01 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Radioactivity, Staining, Microscopy, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 6. Role of endothelin-1 in stretch-dependent BNP promoter ac- tivation and HBEGF ectoshedding. Panels A and C, cardiac myocytes were co-transfected with 0.5 g of 1595 hu- man BNP-luciferase and 0.05 g of actin -galactosidase. 18–24 h post-transfec- tion, cells were deprived of serum for 24 h and then pretreated with the ETA-recep- tor antagonist BQ-610 (1 M; panel A) or the EGFR inhibitor AG-1478 (250 nM; panel C) for 1 h. Myocytes were then sub- jected to a static or strain environment (panel A) or treated with endothelin-1 (107 M; panel C) for 48 h, processed, and assayed for luciferase activity (n 3). Panel B, cells were pretreated with BQ- 610 (1 M) for 1 h and then subjected to a static or stretch environment for 0–5 min. Medium was collected and concentrated, and HBEGF was detected by immunopre- cipitation and immunoblotting (n 3). Data are expressed as means S.E. **, p 0.01; ***, p 0.001 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 7. Metalloproteinase activity is required for strain-de- pendent effects. Panel A, cardiac myocytes were pretreated with a metalloproteinase inhibitor, GM-6001 (4 M), for 1 h and then subjected to a static or strain environment for 0–5 min. Medium was collected and concentrated, and HBEGF was detected by immunoprecipitation and immunoblotting (n 3). Panel B, cardiac myocytes were co-transfected with 0.5 g of 1595 hBNP-luciferase and 0.05 g of actin -galacto- sidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and pretreated with the GM-6001 (4 M) or vehicle for 1 h. Myocytes were then subjected to a static or strain environment for 48 h, pro- cessed, and assayed for luciferase activity (n 3). Data are expressed as means S.E. **, p 0.01 versus static control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Transfection, Luciferase, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 8. Role of ROS in strain- dependent effects. Panel A, antioxidant blocks strain-dependent HBEGF ectoshedding. Cardiac myocytes were pre- treated with an antioxidant, N-acetylcys- teine (NAc; 10 mM), for 1 h and then sub- jected to a static or strain environment for 0–5 min. Medium was collected and con- centrated, and HBEGF was detected by immunoprecipitation and immunoblot- ting. Panels B–D, effect of antioxidant on strain-dependent (panel B) and ET-1-de- pendent (panel C) BNP promoter activa- tion or on strain-dependent ET-1 pro- moter activation (panel D). Cardiac myocytes were co-transfected with 0.5 g of 1595 hBNP-luciferase or 1315 rat ET-1-luciferase and 0.05 g of actin -ga- lactosidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and then pretreated NAc (10 mM) for 1 h. Myo- cytes were subjected to a static or strain environment (panels B and D) or endothe- lin-1 (107 M; panel C) for 48 h, processed, and assayed for luciferase activity. Data are expressed as means S.E. (n 3). NS, not significant; *, p 0.05; **, p 0.01 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Transfection, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 9. Role of NAD(P)H oxidase in strain-dependent effects. Panel A, Effect of NAD(P)H oxidase inhibition on strain-dependent HBEGF ectoshedding. Cardiac myocytes were pretreated with the NAD(P)H inhibitor and apocynin (100 M) for 1 h and then subjected to a static or strain environment for 0–5 min. Medium was collected and concentrated. HBEGF was detected by immunoprecipitation and immunoblotting. Panels B–D, role of NAD(P)H oxidase in strain-dependent (panel B) and ET-1-dependent (panel C) BNP promoter activation or on strain-dependent ET-1 promoter activation (panel D). Cardiac myocytes were co-transfected with 0.5 g of 1595 hBNP-luciferase or 1315 rat ET-1-luciferase and 0.05 g of actin -galactosidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and then pretreated with apocynin (100 M) for 1 h. Myocytes were subjected to a static or strain environment (panels B and D) or endothelin-1 (107 M; panel C) for 48 h, processed, and assayed for luciferase activity. Data are expressed as means S.E. (n 3). NS, not significant; *, p 0.05; **, p 0.01 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Immunoprecipitation, Western Blot, Activation Assay, Transfection, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 10. Schematic representation of signaling cascade linking mechanical strain to activation of the BNP gene promoter. Strain activates NAD(P)H oxidase, which generates ROS. This pro- motes production of ET-1, which stimulates metalloproteinase-medi- ated cleavage of pro-HBEGF to HBEGF, activates the EGFR and sub- sequently leads to an increase in hBNP promoter activity.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Activity Assay