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OriGene human epha2 cdna

Fig. 1. Sketch layout of PIE-FCCS and its role in membrane protien interaction. (A) Diagram illustrates the optical path of dual-color PIE-FCCS. A super-continuum, pulsed laser-generated excitation beams (488 and 561 nm) that were directed into the sample by a dichroic mirror and microscope objective. The lasers were focused to a diameter of about 250 nm on the plasma membrane of live cells. Fluorescence emission was collected by the objective, filtered, and directed to single-photon counting modules con- nected to a TCSPC module for data recording. (B) Epifluorescence images are shown for COS-7 cells expressing EGFR-mCH (top) and <t>EphA2-eGFP</t> (bottom). (C) Summary data from independent experiments collected on five different days of single-cell measurements are shown for the controls and EGFR with EphA2 (***P < 0.0004). (D to F) Repre- sentative single-cell PIE-FCCS data are shown for a monomer control (SRC), dimer control (GCN4), and EGFR with EphA2 in COS-7 cells, respectively. In each graph, the green line is the autocorrelation function (ACF) for eGFP, the red line is the ACF for mCH, and the blue line is the cross-correlation function (CCF) between both protein constructs.

Human Epha2 Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes."

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

Journal: Science advances

doi: 10.1126/sciadv.adl0649

Figure Legend Snippet: Fig. 1. Sketch layout of PIE-FCCS and its role in membrane protien interaction. (A) Diagram illustrates the optical path of dual-color PIE-FCCS. A super-continuum, pulsed laser-generated excitation beams (488 and 561 nm) that were directed into the sample by a dichroic mirror and microscope objective. The lasers were focused to a diameter of about 250 nm on the plasma membrane of live cells. Fluorescence emission was collected by the objective, filtered, and directed to single-photon counting modules con- nected to a TCSPC module for data recording. (B) Epifluorescence images are shown for COS-7 cells expressing EGFR-mCH (top) and EphA2-eGFP (bottom). (C) Summary data from independent experiments collected on five different days of single-cell measurements are shown for the controls and EGFR with EphA2 (***P < 0.0004). (D to F) Repre- sentative single-cell PIE-FCCS data are shown for a monomer control (SRC), dimer control (GCN4), and EGFR with EphA2 in COS-7 cells, respectively. In each graph, the green line is the autocorrelation function (ACF) for eGFP, the red line is the ACF for mCH, and the blue line is the cross-correlation function (CCF) between both protein constructs.

Techniques Used: Membrane, Generated, Microscopy, Clinical Proteomics, Fluorescence, Expressing, Control, Construct

Fig. 2. PIE-FCCS investigation of the homomeric interactions of EGFR and EphA2. (A) Self-assembly of EGFR was studied under various conditions. PIE-FCCS data were recorded, and the resulting fc values are reported before treatment (white), after the addition of EGF (gray), after treatment with the PLC inhibitor U73122 (blue), and after treatment with the PLC activator 3M3FBS (green). (B) Diffusion coefficients for EGFR are shown for each treatment condition. (C) Epifluorescence images of EGFR express- ing COS-7 cells without (top) and with (bottom) EGF ligand treatment. (D) Homomultimerization state of EphA2 was recorded under the same treatments except the li- gand treatment was with EA1. (E) Diffusion coefficients are shown for EphA2 under each treatment condition. (F) Epifluorescence images of EphA2 expressing COS-7 cells are shown without (top) and with (bottom) EA1 ligand treatment. In the box-and-whiskers plots, the whiskers indicate the maximum and minimum values; the box indi- cates the 25th to 75th percentile, and the line is the median value (median value shown as text). We performed one-way analysis of variance (ANOVA) tests with uncor- rected Fisher’s least significant difference (LSD) post hoc tests to obtain adjusted and individual P values (*P < 0.0367, **P < 0.0034, and ****P < 0.0001). Diffusion coefficient data are represented as mean values ± SEM. NS, not significant.

Figure Legend Snippet: Fig. 2. PIE-FCCS investigation of the homomeric interactions of EGFR and EphA2. (A) Self-assembly of EGFR was studied under various conditions. PIE-FCCS data were recorded, and the resulting fc values are reported before treatment (white), after the addition of EGF (gray), after treatment with the PLC inhibitor U73122 (blue), and after treatment with the PLC activator 3M3FBS (green). (B) Diffusion coefficients for EGFR are shown for each treatment condition. (C) Epifluorescence images of EGFR express- ing COS-7 cells without (top) and with (bottom) EGF ligand treatment. (D) Homomultimerization state of EphA2 was recorded under the same treatments except the li- gand treatment was with EA1. (E) Diffusion coefficients are shown for EphA2 under each treatment condition. (F) Epifluorescence images of EphA2 expressing COS-7 cells are shown without (top) and with (bottom) EA1 ligand treatment. In the box-and-whiskers plots, the whiskers indicate the maximum and minimum values; the box indi- cates the 25th to 75th percentile, and the line is the median value (median value shown as text). We performed one-way analysis of variance (ANOVA) tests with uncor- rected Fisher’s least significant difference (LSD) post hoc tests to obtain adjusted and individual P values (*P < 0.0367, **P < 0.0034, and ****P < 0.0001). Diffusion coefficient data are represented as mean values ± SEM. NS, not significant.

Techniques Used: Diffusion-based Assay, Expressing

Fig. 3. Heteromultimerization of EGFR and EphA2 in the presence of ligands and PLC drugs. (A) PIE-FCCS data for EGFR-mCH and EphA2-eGFP coexpressed in COS-7 cells. Data were recorded before treatment (white), after the addition of EGF (gray) or EA1 (orange), and after treatment with the PLC inhibitor U73122 (blue) or with the PLC activator 3M3FBS (green). (B) Diffusion coefficients of EGFR-mCH after treatment with ligands and PLC-regulating drugs. (C) Diffusion coefficients of EphA2-eGFP after treatment with ligands and PLC-regulating drugs. (D) Representative epifluorescence images from each set of experiments. One-way ANOVA tests with uncorrected Fisher’s LSD post hoc tests show the individual P values (*P < 0.0259, **P < 0.003, and ****P < 0.0001). Diffusion coefficient data are represented as mean values ± SEM.

Figure Legend Snippet: Fig. 3. Heteromultimerization of EGFR and EphA2 in the presence of ligands and PLC drugs. (A) PIE-FCCS data for EGFR-mCH and EphA2-eGFP coexpressed in COS-7 cells. Data were recorded before treatment (white), after the addition of EGF (gray) or EA1 (orange), and after treatment with the PLC inhibitor U73122 (blue) or with the PLC activator 3M3FBS (green). (B) Diffusion coefficients of EGFR-mCH after treatment with ligands and PLC-regulating drugs. (C) Diffusion coefficients of EphA2-eGFP after treatment with ligands and PLC-regulating drugs. (D) Representative epifluorescence images from each set of experiments. One-way ANOVA tests with uncorrected Fisher’s LSD post hoc tests show the individual P values (*P < 0.0259, **P < 0.003, and ****P < 0.0001). Diffusion coefficient data are represented as mean values ± SEM.

Techniques Used: Diffusion-based Assay

Fig. 4. EGFR and EphA2 phosphorylation changes after treatment with ligand and PLC drugs. (A) Representative Western blots after treatment with EA1 (15 min, 500 ng/ml), 3M3FBS (45 min, 25 μM), or 3M3FBS followed by EA1. (B) Representative Western blots after treatment with U73122 (15 min, 5 μM), EA1 (as above), EGF (15 min, 100 ng/ml), or U73122 followed by each ligand. (C) Quantification of EphA2 pS897 levels in all conditions normalized to the corresponding total EphA2 bands. N = 6 to 12. Statistical analysis was performed using a Kruskal-Wallis test [H(7) = 19.45, P = 0.007] with a Mann-Whitney U test for comparisons between groups. Signifi- cance values were adjusted by the Bonferroni correction for multiple tests. (D) Quantification of total EphA2 levels in all conditions normalized to actin. N = 6 to 12. Sta- tistical analysis was performed using a Kruskal-Wallis test [H(7) = 38.44, P = 3.0 × 10−6] with a Mann-Whitney U test for comparisons between groups. Significance values were adjusted by the Bonferroni correction for multiple tests. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure Legend Snippet: Fig. 4. EGFR and EphA2 phosphorylation changes after treatment with ligand and PLC drugs. (A) Representative Western blots after treatment with EA1 (15 min, 500 ng/ml), 3M3FBS (45 min, 25 μM), or 3M3FBS followed by EA1. (B) Representative Western blots after treatment with U73122 (15 min, 5 μM), EA1 (as above), EGF (15 min, 100 ng/ml), or U73122 followed by each ligand. (C) Quantification of EphA2 pS897 levels in all conditions normalized to the corresponding total EphA2 bands. N = 6 to 12. Statistical analysis was performed using a Kruskal-Wallis test [H(7) = 19.45, P = 0.007] with a Mann-Whitney U test for comparisons between groups. Signifi- cance values were adjusted by the Bonferroni correction for multiple tests. (D) Quantification of total EphA2 levels in all conditions normalized to actin. N = 6 to 12. Sta- tistical analysis was performed using a Kruskal-Wallis test [H(7) = 38.44, P = 3.0 × 10−6] with a Mann-Whitney U test for comparisons between groups. Significance values were adjusted by the Bonferroni correction for multiple tests. *P < 0.05, **P < 0.01, and ***P < 0.001.

Techniques Used: Phospho-proteomics, Western Blot, MANN-WHITNEY

Fig. 5. AlFoM model of the heterodimer of EGFR residues 669 to 1210 (cyan) and EphA2 residues 559 to 976 (purple). Predictions are shown for EphA2 WT (A) or for the S897E/S901E mutant (B) representative of phosphorylation at serine residues (red bubbles). Yellow residues indicate the active site of each kinase region. The purple halo encompasses the EphA2 SAM domain, while the cyan halo shows the EGFR JM domain. The red arrow indicates the EphA2 kinase active site, which is steri- cally blocked by the EGFR kinase domain in (A), but not in (B).

Figure Legend Snippet: Fig. 5. AlFoM model of the heterodimer of EGFR residues 669 to 1210 (cyan) and EphA2 residues 559 to 976 (purple). Predictions are shown for EphA2 WT (A) or for the S897E/S901E mutant (B) representative of phosphorylation at serine residues (red bubbles). Yellow residues indicate the active site of each kinase region. The purple halo encompasses the EphA2 SAM domain, while the cyan halo shows the EGFR JM domain. The red arrow indicates the EphA2 kinase active site, which is steri- cally blocked by the EGFR kinase domain in (A), but not in (B).

Techniques Used: Mutagenesis, Phospho-proteomics

Fig. 6. Comparison of EGFR and EphA2 interactions with the lipid bilayer. (A) Snapshots illustrating the predominant conformation of the EGFR-EphA2 heterodimer after molecular dynamics simulations. The intracellular view highlights the EGFR-EphA2 heterodimer’s association with the lipid bilayer, where EGFR is shown in salmon and EphA2 in purple-blue surfaces. Cholesterol, PIP2, and POPS are depicted as cyan, green, and yellow spheres, respectively, while POPC is displayed as gray lines for clarity. The image on the right shows the interaction interface surface of the heterodimer viewed from the intracellular side. (B) Comparison of the number of contacts between EGFR and EphA2 with PIP2 in the membrane over the course of the simulation. The number of contacts is averaged across four replica simulations, considering a 6-Å cutoff distance. KD, kinase domain; SAM, sterile alpha motif.

Figure Legend Snippet: Fig. 6. Comparison of EGFR and EphA2 interactions with the lipid bilayer. (A) Snapshots illustrating the predominant conformation of the EGFR-EphA2 heterodimer after molecular dynamics simulations. The intracellular view highlights the EGFR-EphA2 heterodimer’s association with the lipid bilayer, where EGFR is shown in salmon and EphA2 in purple-blue surfaces. Cholesterol, PIP2, and POPS are depicted as cyan, green, and yellow spheres, respectively, while POPC is displayed as gray lines for clarity. The image on the right shows the interaction interface surface of the heterodimer viewed from the intracellular side. (B) Comparison of the number of contacts between EGFR and EphA2 with PIP2 in the membrane over the course of the simulation. The number of contacts is averaged across four replica simulations, considering a 6-Å cutoff distance. KD, kinase domain; SAM, sterile alpha motif.

Techniques Used: Comparison, Membrane, Sterility

Fig. 7. Model for EGFR-EphA2 heterodimerization. When PIP2 levels are normal, EGFR (blue) and EphA2 (orange) do not interact in the absence of ligand. When EGF is present, active heterodimer forms in which EGFR Y1068 and EphA2 S897 are phosphorylated. The active conformation of the ICDs allows a more open EphA2 kinase domain with the EGFR JM free to move away from the membrane. Under increased PIP2 conditions, PIP2 promotes inactive heterodimer formation. The inac- tive dimer is likely stabilized by the EGFR JM electrostatically interacting with the membrane, and the EGFR ICD is pulled toward the membrane to close the EphA2 kinase domain.

Figure Legend Snippet: Fig. 7. Model for EGFR-EphA2 heterodimerization. When PIP2 levels are normal, EGFR (blue) and EphA2 (orange) do not interact in the absence of ligand. When EGF is present, active heterodimer forms in which EGFR Y1068 and EphA2 S897 are phosphorylated. The active conformation of the ICDs allows a more open EphA2 kinase domain with the EGFR JM free to move away from the membrane. Under increased PIP2 conditions, PIP2 promotes inactive heterodimer formation. The inac- tive dimer is likely stabilized by the EGFR JM electrostatically interacting with the membrane, and the EGFR ICD is pulled toward the membrane to close the EphA2 kinase domain.

Techniques Used: Membrane

Polymerase Chain Reaction:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: Overexpression of EphA2 WT and EphA2 Fibronectin III Domain (G391R) Mutant For the purpose of overexpressing EphA2 in the BEAS-2B cell line, which is a human immortalized bronchial epithelial cell line, we produced a plasmid containing the EphA2 open reading frame by TOPO-cloning methodology (Invitrogen). .. After obtaining human EphA2 cDNA (Origene), it was PCR-amplified and cloned into the pCDNA6.2 vector (Invitrogen), which contains a C-terminal V5 tag as well as antibiotic resistance genes for selection of stable transfectants in mammalian cells. ..

Clone Assay:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: .. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. ..

Plasmid Preparation:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: .. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. ..

Selection:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: .. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. ..

Mutagenesis:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: .. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. ..

Transfection:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: .. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. ..

Cell Culture:

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation
Article Snippet: .. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. ..

Review