egft  (MedChemExpress)

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A Protein analysis of the different purification steps by Coomassie blue-stained SDS-PAGE. Molecular weights (M); Lane 1: commercial recombinant hEGF. Lane 2: supernatant of the E.coli fermentor culture before concentration. Lane 3: 30x concentrated supernatant by tangential flow filtration. Lane 4: product of the first purification step (anionic exchange chromatography). Lane 5: purified product after the final purification step (gel filtration chromatography). B Determination of the molecular weight of hEGF and EGFt by mass spectrometry (MALDI-TOF). The analysis confirmed the corresponding molecular weight of the hEGF (6216.6 Da) and EGFt (5087.9 Da). C Analysis of the state of folding of purified hEGF and EGFt by RP-HPLC. The peaks on the chromatogram correspond to the elution time of well-folded hEGF.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Purification, Staining, SDS Page, Recombinant, Concentration Assay, Filtration, Chromatography, Molecular Weight, Mass Spectrometry

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: Cell lysate from MDA-MB-468 cells were treated with the indicated concentrations of hEGF, EGFt or a mixture of both for 30 min. Then the samples were cross-linked by addition of 40 mM of glutaraldehyde and analyzed by Western blotting using an anti-EGFR antibody. The position of the EGFR monomers and dimmers is indicated.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A Analysis of the total phosphorylation of EGFR. MDA-MB-468 cells were treated with 3 nM, 150 nM hEGF or 150 nM EGFt for 10 min. The whole cell lysates were analyzed in parallel by Western blotting with an antibody against total-phosphotyrosine residues (PY20) and with an antibody against EGFR (EGFR 1005). B Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the internalization of the receptor. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM nM EGFt for the indicated periods of time. The whole cell lysates were analyzed by Western blotting with site-specific antibodies for phospho-EGFR tyrosine 1045 and serines 1046/47. C Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the proliferation signaling pathway. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM EGFt for the indicated periods of time. The whole cell lysates were analysed by Western blotting with site-specific antibodies for phospho-EGFR tyrosines 1068 and 1173. Untreated cells were used as a negative control (Ctl) and β-actin levels were used as the loading control in Western blotting.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Phospho-proteomics, Western Blot, Negative Control, Control

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A MCF-7 and Caco-2 cells were treated with 3 nM, 150 nM hEGF or 150 nM EGFt for 30 min at 4°C and then incubated at 37°C for 15 min. The detection of EGFR in the cell membrane was determined by performing a cell-ELISA assay with specific antibodies. Each column in the graph represents the relative EGFR expression in the cell membrane versus untreated control cells (CTL) and was the mean ± SEM of three independent experiments (*P<0.05 vs. control cells). B MDA-MB-468 cells were exposed for various times to 150 nM hEGF or 150 nM EGFt and stained for EGFR using FITC anti-EGFR antibody (green). The nucleus and its membrane were stained using Hoescht (blue) and Cy3 anti-lamin B1 antibody (red), respectively. Confocal images were acquired. C The merge images corresponding to 180 min of treatment were magnified and some slices of a merged xz reconstruction of the stack (slices at 0.3 microns on z axis) are shown. Arrows indicate green signals within the cell nucleus. Scale bar, 10 μm.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Incubation, Membrane, Enzyme-linked Immunosorbent Assay, Expressing, Control, Staining

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A MCF-7 and Caco-2 cycloheximide treated cells were incubated with 3 nM, 150 nM hEGF or 150 nM EGFt at 37°C for different time periods as indicated. Cells were lysed, and the amount of EGFR was determined by Western blotting. Actin levels were used as loading control. B Levels of EGFR were determined by densitometry and normalized versus actin levels. Each column represents the mean of two replicates ± SEM.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Incubation, Western Blot, Control

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: MCF-7 and Caco-2 cells were incubated for 72 h or 96 h h in a culture medium without FBS supplemented with 10, 20 and 150 nM hEGF or EGFt. The cell proliferation was assessed by MTT assays. Each column in the graph represents the relative cell proliferation versus untreated cells (control) and was the mean ± SEM of three independent experiments (*P<0.05 vs. control cells).

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Incubation, Control

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A Protein analysis of the different purification steps by Coomassie blue-stained SDS-PAGE. Molecular weights (M); Lane 1: commercial recombinant hEGF. Lane 2: supernatant of the E.coli fermentor culture before concentration. Lane 3: 30x concentrated supernatant by tangential flow filtration. Lane 4: product of the first purification step (anionic exchange chromatography). Lane 5: purified product after the final purification step (gel filtration chromatography). B Determination of the molecular weight of hEGF and EGFt by mass spectrometry (MALDI-TOF). The analysis confirmed the corresponding molecular weight of the hEGF (6216.6 Da) and EGFt (5087.9 Da). C Analysis of the state of folding of purified hEGF and EGFt by RP-HPLC. The peaks on the chromatogram correspond to the elution time of well-folded hEGF.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Purification, Staining, SDS Page, Recombinant, Concentration Assay, Filtration, Chromatography, Molecular Weight, Mass Spectrometry

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: Cell lysate from MDA-MB-468 cells were treated with the indicated concentrations of hEGF, EGFt or a mixture of both for 30 min. Then the samples were cross-linked by addition of 40 mM of glutaraldehyde and analyzed by Western blotting using an anti-EGFR antibody. The position of the EGFR monomers and dimmers is indicated.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A Analysis of the total phosphorylation of EGFR. MDA-MB-468 cells were treated with 3 nM, 150 nM hEGF or 150 nM EGFt for 10 min. The whole cell lysates were analyzed in parallel by Western blotting with an antibody against total-phosphotyrosine residues (PY20) and with an antibody against EGFR (EGFR 1005). B Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the internalization of the receptor. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM nM EGFt for the indicated periods of time. The whole cell lysates were analyzed by Western blotting with site-specific antibodies for phospho-EGFR tyrosine 1045 and serines 1046/47. C Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the proliferation signaling pathway. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM EGFt for the indicated periods of time. The whole cell lysates were analysed by Western blotting with site-specific antibodies for phospho-EGFR tyrosines 1068 and 1173. Untreated cells were used as a negative control (Ctl) and β-actin levels were used as the loading control in Western blotting.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Phospho-proteomics, Western Blot, Negative Control, Control

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A MCF-7 and Caco-2 cells were treated with 3 nM, 150 nM hEGF or 150 nM EGFt for 30 min at 4°C and then incubated at 37°C for 15 min. The detection of EGFR in the cell membrane was determined by performing a cell-ELISA assay with specific antibodies. Each column in the graph represents the relative EGFR expression in the cell membrane versus untreated control cells (CTL) and was the mean ± SEM of three independent experiments (*P<0.05 vs. control cells). B MDA-MB-468 cells were exposed for various times to 150 nM hEGF or 150 nM EGFt and stained for EGFR using FITC anti-EGFR antibody (green). The nucleus and its membrane were stained using Hoescht (blue) and Cy3 anti-lamin B1 antibody (red), respectively. Confocal images were acquired. C The merge images corresponding to 180 min of treatment were magnified and some slices of a merged xz reconstruction of the stack (slices at 0.3 microns on z axis) are shown. Arrows indicate green signals within the cell nucleus. Scale bar, 10 μm.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Incubation, Membrane, Enzyme-linked Immunosorbent Assay, Expressing, Control, Staining

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: A MCF-7 and Caco-2 cycloheximide treated cells were incubated with 3 nM, 150 nM hEGF or 150 nM EGFt at 37°C for different time periods as indicated. Cells were lysed, and the amount of EGFR was determined by Western blotting. Actin levels were used as loading control. B Levels of EGFR were determined by densitometry and normalized versus actin levels. Each column represents the mean of two replicates ± SEM.

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Incubation, Western Blot, Control

Journal: PLoS ONE

Article Title: Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity

doi: 10.1371/journal.pone.0069325

Figure Lengend Snippet: MCF-7 and Caco-2 cells were incubated for 72 h or 96 h h in a culture medium without FBS supplemented with 10, 20 and 150 nM hEGF or EGFt. The cell proliferation was assessed by MTT assays. Each column in the graph represents the relative cell proliferation versus untreated cells (control) and was the mean ± SEM of three independent experiments (*P<0.05 vs. control cells).

Article Snippet: hEGF and EGFt encoding sequences were synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Incubation, Control