Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: In vitro characterization of IgG25 and IgG28. (a) FACS-based whole cell binding assay was performed with IgG25 and IgG28 on human MiaPaCa2 and (b) on mouse MC38-CEA cells to determine the apparent Kd. Mean Fluorescence Intensities (MFIs) obtained with IgG25 and IgG28 over the logarithm of their molar concentration (LogM) are reported. (c) Binding competition experiments of IgG25 and IgG28 with ephrinA1 on MiaPaCa2 cells, a control isotypic IgG1 antibody (Ctrl IgG) was used as negative control. IgG25 (filled circles), IgG28 (empty circles), Ctrl IgG (filled triangles). (d) EphA2 immunoprecipitation from lysates of cells treated with Ctrl IgG, IgG25, IgG28 and ephA1-Fc, followed by Western Blot with antiphosphotyrosine antibody. After stripping, the same filter was probed with EphA2 antibody as loading control. (e) FACS-based whole cell binding with IgG25 and IgG28 on mouse N2A cells transiently transfected with expression vectors coding for members of Eph A receptor family (EphA1, EphA2, EphA3, EphA4, EphA5, and EphA7).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vitro, Cell Binding Assay, Fluorescence, Concentration Assay, Binding Assay, Control, Negative Control, Immunoprecipitation, Western Blot, Stripping Membranes, Transfection, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25- and IgG28-mediated EphA2 internalization and degradation in MiaPaCa2 cells. (a) EphA2 internalization in response to IgG25, IgG28, and ephrinA1-Fc treatment. mAbs and ephrinA1-Fc labeled in red while cell nuclei in blue. Images were acquired at 20× magnification. Localization was revealed 1 hour after incubation on cells either at 4°C or at 37°C. (b) Time course Western blot analysis of EphA2 degradation after treatment with control IgG, IgG25, IgG28, and ephrinA1-Fc; anti- β -actin was used as loading control. (c) Densitometric analysis of the level of EphA2 expression measured by Western Blot in cells treated with control IgG (asterisks), IgG25 (squares), IgG28 (triangles), and ephrinA1-Fc (circles). Data are expressed as percentage of EphA2 expression over time.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Labeling, Incubation, Western Blot, Control, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 downstream signaling in MiaPaCa2 cells treated with IgG25 and IgG28. Cell lysates of MiaPaCa2 were assayed by Western Blotting with antiphospho-Akt, antiphospho-ERK, and anti-phospho FAK (Tyr 576). Antibodies directed to total Akt, total FAK, and actin were used as loading controls.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Western Blot

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: Effect of EphA2 antibodies on angiogenesis. (a) Representative CD31 immunostaining of paraffin embedded tumor section (magnification bar = 20 m). (b) Quantitative analysis of CD31 staining in tumors treated with control IgG, IgG25, and IgG28. The average data obtained from the analyses of two tumors selected from each group are reported as the percentage of CD31 + area in each entire section. In all panels, asterisks indicate statistically significant differences with respect to the control group (Student's t -test; P <.05).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Immunostaining, Staining, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 protein expression in tumors from treated mice. (a) Western Blot analysis with anti-EphA2 antibodies of lysates from five tumors treated with control IgG, with IgG25, or six tumors treated with IgG28. (b) Densitometric analysis of the ratio between EphA2 levels revealed by anti-EphA2 antibody in nonsaturating conditions and actin expression measured with antiactin antibody. Data on y axis are expressed as arbitrary units.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25 and IgG28 modulate in vivo EphA2 downstream signaling: Western Blot analyses of tumor lysates with antiphospho FAK (Tyr 576) and anti-phospho Akt. Antibodies to total Akt, total FAK, and actin were used as loading controls. P values were calculated with respect to average densitometric value of group treated with control IgG.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vivo, Western Blot, Control

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: Q-RT-PCR primer list

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques:

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: EPH and EFN plasmid list

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Plasmid Preparation

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: Epha2 deficiency reduces inflammatory responses induced by flagellin or P. aeruginosa in mouse lungs (A) EPHA2 protein expression in tissues recovered from WT and Epha2 KO mice was analyzed by WB. Ponceau staining was used as a `loading control. (B–E) WT and Epha2 KO mice were infected with 1 × 10 9 CFU live P. aeruginosa via intranasal administration. The inflammatory state was evaluated in BALF and lung tissue 6 h later. (B) HE-staining image of lung tissue. The scale bar represents 10 μm. (C) Quantitative analysis of the number of cell nuclei per tissue area (μm 2 ). n = 4, 4, 4, 6 (from left to right, two-way ANOVA, Tukey’s test). (D) ELISA of KC in BALF. n = 4, 4, 3, 5 (from the left lane). (two-way ANOVA, Tukey’s test). (E) RT-qPCR analysis of inflammatory cytokine and EphaA2 mRNA expression levels in lung tissue. All primer information used in this study is listed in . n = 4, 4, 3, 6 (from left to right, KC, Il-6, Il-1β: two-way ANOVA, Tukey’s test, Epha2: unpaired multiple t -test, Bonferroni-Dunn test). (F and G) WT, Epha2 +/−, and KO mice were administered 1 μg of FLA-PA via intra-tracheal injection. The respiratory inflammatory state was evaluated by using BALF and lung tissue samples collected 6 h after FLA-PA treatment. (F) ELISA of KC in BALF. n = 4, 7, 4, 3, 3, 4 (from left to right, unpaired t -test, two-tailed). (G) RT-qPCR Analysis of mRNA expression levels in lung tissue. KC and IL-6: n = 5, 9, 4, 4, 3, 6, Il-1β: n = 4, 6, 4, 4, 3, 6, Epha2: n = 4, 4, 4, 3, 3, 3 (from left to right, KC, Il-6, Il-1β: unpaired t -test, two-tailed, Epha2: unpaired multiple t -test, Bonferroni-Dunn test). Data are presented as mean ± SEM (C, D, E, F, G).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Expressing, Staining, Control, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Injection, Two Tailed Test

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: Gene silencing of EPHA2 attenuates the inflammatory response to PAMPs in AECs (A) The RT-qPCR analysis of EPH and EFN family mRNA expression in BEAS-2B cells ( n = 3). (B and C) BEAS-2B parental cells were transfected with 20 nM EPHA2 siRNA and treated with indicated 1 μg/mL LPS-SE, 1 μg/mL PamCSK4, 1 μg/mL poly (I:C) and 100 ng/mL FLA-PA for 4 h. IL-8 and IL-6 mRNA expression was analyzed by RT-qPCR. (B) Confirmation of EPHA2 KD efficiency by siRNA ( n = 4, p value: vs. siNT, unpaired multiple t -test, Bonferroni-Dunn test). (C) IL-8 and IL-6 mRNA expression analysis. LPS and Pam: n = 4, poly (I:C): n = 3, FLA-PA: siNT; n = 5, siEPHA2; n = 3. p value: vs. siNT-PAMPs, RM two-way ANOVA, Tukey’s test. (D) EPHA2 protein expression in WT and EPHA2 KO BEAS-2B cell lines. Na + /K + ATPase was used as a loading control. (E and F) IL-8 and IL-6 mRNA expression was analyzed by RT-qPCR 4 h after treatment of 100 ng/mL FLA-PA (E) or MOI 10 live P. aeruginosa (F) in WT and EPHA2 KO BEAS-2B cells. n = 3, p value: vs. WT-PAMPs, RM two-way ANOVA, Tukey’s test. (G) BEAS-2B cells were transfected with 20 nM siRNA (siEPHA2, siEFNA1, or both) and treated with 100 ng/mL FLA-PA for 4 h. The expression of the inflammatory cytokines, EPHA2 and EFNA1, was analyzed using RT-qPCR. siNT; n = 5, siEPHA2; n = 3, siEFNA1; n = 3, siEPHA2+EFNA1; n = 4, RM two-way ANOVA, Tukey’s test. Data are presented as mean ± SEM (A, B, C, E, F, G).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: Establishment of EPH-EFN trans -binding real-time measurement in live AECs (A) Schematic of established iLgBiT-EPH-HA and SmBiT-Flag-EFN(-V5) constructs. Original plasmid information of EPH and EFN genes used in this study is listed in . SP: signal peptide, LBD: ligand binding domain, FNIII: fibronectin-type III, TM: transmembrane domain, SAM: sterileαmotif, PDZ: PDZ-binding, RBD: receptor binding domain, GPI-anc: GPI anchor propeptide region. (B) Western blotting analysis of iLg-EPH-HA and Sm-Flag-EFN protein expression in BEAS-2B cells with stable expression. iLg-EPH-HA expression cells were treated with 1 μg/mL Dox for 72 h to induce their expression. The exogenous EPHs and EFNs were detected by anti-HA antibody and anti-Flag antibody, respectively. The expected molecular weight (MW: kDa) were shown below each band. (C) Plasma membrane expression analysis of iLg-EPH-HA in BEAS-2B cells by using HiBiT-Halo protein. iLg-EPH-HA expression was induced by treatment of Dox (A2, A4: 50 ng/mL, B1: 500 ng/mL, B3: 150 ng/mL, B4, B6: 1000 ng/mL) for 48 h ( n = 3). (D) PM expression of Sm-Flag-EFN in BEAS-2B cells was measured by cell-surface ELISA using anti-Flag antibody and HRP-conjugated anti-mouse antibody ( n = 3). (E) Schematic of EPH-EFN trans -binding measurement using iLg-EPH-HA-expressing cells (EPH, black) and Sm-Flag-EFN-expressing cells (EFN, red) co-culturing system. EPH-EFN binding is formed in cytonemes contact site between EPH or EFN expressing cells, and the signal of complemented NanoLuc reflects the amount of EPH-EFN interaction. Membrane-permeable substrate enables to detect EPH-EFN interactions inside cells. (F) Immunostaining of the cell-cell contact site during the co-culturing of iLg-EPHA2-HA and Sm-Flag-EFNA1 or iLg-EPHA4-HA and Sm-Flag-EFNB2-V5 expressing cells. The iLg-EPH-HA were labeled in green and the Sm-Flag-EFN were labeled in red. Outline of iLg-EPHA4-HA expressing cell (Green) or Sm-Flag-EFNB2-V5 (Red) expressing cells was shown in broken line. The right panel shows an enlarged image of the white box area of left panel, and white arrowhead shows co-localization. (G) The quantitative results of EPH-EFN trans -binding measurement between iLg-EPH and Sm-Flag-EFN in BEAS-2B cells were displayed as a heatmap, with the trans -binding amount relative to EPHA2-EFNA1 pair ( n = 3). Data are presented as mean ± SEM (C, D).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Binding Assay, Construct, Plasmid Preparation, Ligand Binding Assay, Western Blot, Expressing, Molecular Weight, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Immunostaining, Labeling

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: The impact of pathogen components on the trans -binding of EPH and EFN (A) Real-time EPH-EFN trans -binding measurement was performed during the treatment of PAMPs and heat-killed bacteria. We presented relative changes in EPH-EFN binding compared to the control (CON) at the 4-h after stimulation in a heatmap. All the experimental results over the entire time course are presented in Figure S4 ( n = 3). (B) Time course analysis of the changes in EPHA2-EFNA1 trans -binding following treatment with specified pathogen molecules. We presented the statistical significance of the results at the 4-h after treatment ( n = 3). RM one-way ANOVA, Dunnett’s test. (C) Comparison of the time course of inflammatory response and the changes in EPHA2-EFNA1 trans -binding during treatment with 100 ng/mL FLA-PA in BEAS-2B cell. The inflammatory response was assessed by analyzing IL-8 mRNA level by RT-qPCR ( n = 3). (D) The correlation between EPHA2-EFNA1 trans -binding ( Figure 4 A) and IL-8 or IL-6 mRNA induction ( Figure S4 B) in BEAS-2B cells 4 h after each stimulation. Data are presented as mean ± SEM (B (+SEM only), C, D).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Binding Assay, Bacteria, Control, Comparison, Quantitative RT-PCR

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: FLA-PA induces TLR5 dependent EFNA1 release from the PM in AECs (A) WT or TLR5 KO BEAS-2B cells were transfected with 20 nM EPHA2 siRNA and the FLA-PA induced inflammatory response was analyzed by RT-qPCR. n = 3, RM two-way ANOVA, Tukey’s test. (B) WT or TLR5 KO BEAS-2B cells were treated with 100 ng/mL FLA-PA for 0 to 120 min, and the total EPHA2, phospho-S897 (pS897) and phosphor-Y772 (pY772) EPHA2 levels were analyzed by WB. Ponceau staining was used as loading control. (C) The ratio of pS897 or pY772/total EPHA2 at 1 h after FLA-PA treatment was calculated from the intensity of the WB result. n = 10, 10, 8, 8 (pS897, from left to right) and n = 7, 7, 6, 6 (pY772, from left to right), unpaired t- test, two-tailed. (D) Analysis of whether the TLR5-dependent dissociation of EPHA2-EFNA1 by FLA-PA results in an effect on EPHA2 or EFNA1. WT or TLR5 KO BEAS-2B cells expressing iLg-EPHA2-HA or Sm-Flag-EFNA1 were co-cultured in indicated combinations. The EPHA2-EFNA1 trans -binding relative to CON was quantified at 150 min after 100 ng/mL FLA-PA treatment. n = 3, unpaired multiple t -test, Bonferroni-Dunn test. (E) The analysis of EFN-RBD dependency in FLA-PA induced EPHA2-EFNA1 dissociation. BEAS-2B cells expressing Sm-Flag fused WT-EFNA1, a5 RBD-EFNA1 or a1 RBD-EFNA5 were co-cultured with iLg-EPHA2-HA-expressing cells. The EPHA2 and WT-EFNA1, a5 RBD-EFNA1 or a1 RBD-EFNA5 trans -binding was measured during 100 ng/mL FLA-PA treatment. The interaction ratio relative to CON was quantified at 180 min after FLA-PA-treatment. n = 3, unpaired multiple t -test, Bonferroni-Dunn test. (F) The FLA-PA impact on EPHA2 p.m. level. iLg-EPHA2-HA expressing BEAS-2B cell were treated with 100 ng/mL FLA-PA for 0–3 h and PM expression level of iLg-EPHA2-HA was analyzed by using HiBiT-Halo protein. n = 3, RM one-way ANOVA, Dunnett’s test. (G) The FLA-PA impact on EFNA1 p.m. level. SmBiT-Flag-EFNA1 p.m. levels in BEAS-2B cells under 100 ng/mL FLA-PA treatment for 0–4 h was analyzed by cell surface ELISA with anti-Flag antibodies. n = 3, RM one-way ANOVA, Dunnett’s test. (H) Proteasomal or lysosomal degradation inhibitor effect on FLA-PA induced EFNA1 loss from PM. Sm-Flag-EFNA1 expressing BEAS-2B cells were treated with 10 μM MG132 or 1 μM Bafilomycin A1 (Baf A1) for 2 h before and during treatment with 100 ng/mL FLA-PA. PM level of Sm-Flag-EFNA1 was measured at 1 h after FLA-PA treatment by cell surface ELISA and shown as relative to CON. n = 3, two-way ANOVA, Tukey’s test. (I and J) FLA-PA effect on endogenous EFNA1 protein level. BEAS-2B parental cells were treated with 100 ng/mL FLA-PA for 4 h, and the endogenous EFNA1 levels in the supernatant (Sup.) and cell lysate (Lysate) were immunoblotted (IB) by using anti-EFNA1 antibody. (J) The intensity of EFNA1 band was quantified. n = 4, unpaired t- test. Data are presented as mean ± SEM (A, C, D, E, F, G, H, J).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Transfection, Quantitative RT-PCR, Staining, Control, Two Tailed Test, Expressing, Cell Culture, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: EFNA1 is cleaved by ADAM9 in response to flagellin-TLR5 signaling (A and B) Schema and result of the released Sm-Flag-EFNA1 measurement. WT or TLR5 KO BEAS-2B cells expressing Sm-Flag-EFNA1 were treated with 100 ng/mL FLA-PA for 4 h, and the supernatant was collected. Then mixture of the collected supernatant containing released Sm-Flag-EFNA1 and NanoBiT substrate were added to iLg-EPHA2-HA expressing cells. The amount of Sm-Flag-EFNA1 in the supernatant was analyzed based on NanoLuc luminescence formed between Sm-Flag-EFNA1 in the supernatant and iLg-EPHA2-HA on the cell surface. n = 3, RM two-way ANOVA, Sidak’s test. (C) The effect of NF-κB inhibitors on FLA-PA-induced EFNA1 release. Sm-Flag-EFNA1 expressing cells were pre-treated with 50 μM Wedelolactone or 10 μM Takinib for 2 h, and then co-treated with 100 ng/mL FLA-PA for 4 h. The supernatant was collected and released Sm-Flag-EFNA1 was measured. n = 3, RM two-way ANOVA, Sidak’s test. (D) The release of EFNA1 in response to various pathogenic stimuli. The measurement of released Sm-Flag-EFNA1 was conducted 4 h after treating with the same concentrations of pathogen components as depicted in Figure 2 n = 3, RM one-way ANOVA, Dunnett’s test. (E) Correlation analysis between the Sm-Flag-EFNA1 release ( Figure 6 D) and the inflammatory cytokine mRNA induction ( Figure S4 B) after 4-h FLA-PA treatment, relative to each control (CON). n = 3. (F and G) The effect of MMP/ADAM inhibitors on FLA-PA-induced EFNA1 release. Sm-Flag-EFNA1 expressing cells were pre-treated with indicated concentration of MMP/ADAM inhibitors for 2 h, and then co-treated with 100 ng/mL FLA-PA for 4 h. (F) The supernatant was collected and used for released Sm-Flag-EFNA1 measurement. DMSO: n = 7, OPN 100 μM: n = 4, Other groups: n = 3, RM two-way ANOVA, Dunnett’s and Sidak’s test. (G) The amount of Sm-Flag-EFNA1 in the supernatant (Sup.) and cell lysate (Lysate) were analyzed by WB by using anti-Flag antibody. (H) Sm-Flag-EFNA1-TEV (SF-a1-TEV) was generated by replacing the metalloprotease recognition sequence of Sm-Flag-EFNA1 (SF-a1) with the TEV protease recognition sequence (Blue). (I and J) The metalloprotease recognition site is crucial for FLA-PA-induced EFNA1 release. BEAS-2B cells expressing SF-a1 and SF-a1-TEV were exposed to FLA-PA for 4 h. The quantities of SF-a1 or SF-a1-TEV released into the supernatant (I) or present on the cell surface (J) were determined using released SF-a1 measurement and cell surface ELISA methods. n = 3, RM two-way ANOVA, Sidak’s test. (K) The impact of MMPs/ADAMs KD on FLA-PA-induced exogenous EFNA1 release. BEAS-2B cells expressing Sm-Flag-EFNA1 were transfected with 20 nM siRNA targeting ADAMs or MMPs, and the amount of released Sm-Flag-EFNA1 was measured 4 h after FLA-PA treatment. siNT: n = 5, Other groups: n = 3, RM two-way ANOVA, Dunnett’s and Sidak’s test. (L) The impact of MMPs/ADAMs KD on FLA-PA-induced endogenous EFNA1 release. BEAS-2B cells were transfected with 20 nM siRNA targeting ADAMs or MMPs, and the amount of EFNA1 in the supernatant (Sup.) and cell lysate (Lysate) 4 h after 100 ng/ml FLA-PA treatment were analyzed by WB. (M) The effect of an ADAM9 KD on FLA-PA-induced EPHA2-EFNA1 dissociation. ADAM9 KD was performed on co-cultured iLg-EPHA2-HA and Sm-Flag-EFNA1 cells, and EPHA2-EFNA1 trans -binding was monitored during FLA-PA treatments at 100 or 200 ng/mL. The graph on the left shows the time-course changes in EPHA2-EFNA1 binding, while the graph on the right presents the statistical analysis of the results obtained 4 h after FLA-PA stimulation. n = 3, RM two-way ANOVA, Dunnett’s test. Data are presented as mean ± SEM (B, C, D, E, F, I, J, K, M (+SEM only)).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Expressing, Control, Concentration Assay, Generated, Sequencing, Enzyme-linked Immunosorbent Assay, Transfection, Cell Culture, Binding Assay

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet: Cleavage of EFNA1 induces inflammatory response in AECs (A) The effect of soluble EFNA1 on cytokine mRNA expression in AECs. BEAS-2B cells were treated with 50 ng/mL EFNA1-Fc for 0–24 h, and the inflammatory response was analyzed by RT-qPCR. n = 3, RM one-way ANOVA, Dunnett’s test. (B) TEV protease-induced specific cleavage of SF-a1-TEV in cells. BEAS-2B cells expressing SF-a1 or SF-a1-TEV were treated with the indicated concentrations of TEV protease in CO2-independent medium and TEV buffer at 37°C for 3 h. The released SF-a1 and SF-a1-TEV in the supernatant were measured. n = 3, RM two-way ANOVA, Sidak’s test. (C) TEV protease-induced specific dissociation of EPHA2 and SF-a1-TEV in cells. iLg-EPHA2-HA expressing cells were co-cultured with SF-a1 or SF-a1-TEV expressing cells, and the EPHA2-EFNA1 trans -binding level was measured during treatment of 200 U/ml TEV protease. n = 3. (D) The impact of specific cleavage of SF-a1-TEV on inflammatory response in AECs. SF-a1 or SF-a1-TEV expressing cells were treated with the indicated concentration of TEV protease for 3 h, and the inflammatory response was analyzed by RT-qPCR. n = 3, two-way ANOVA, Tukey’s test. (E) The effect of MMPs/ADAMs KD on FLA-PA-induced inflammatory cytokine up-regulation. BEAS-2B cells expressing Sm-Flag-EFNA1 were transfected with 20 nM siRNA targeting ADAMs and MMPs, and the inflammatory response was analyzed by RT-qPCR 4 h after treatment with 100 ng/mL FLA-PA. siNT: n = 8, siADAM9: n = 5, Other groups: n = 3, RM two-way ANOVA, Sidak’s test. (F) Correlation analysis between the impact of ADAM/MMP KD on FLA-PA-dependent IL-8 and IL-6 mRNA induction ( Figure 7 E) and EFNA1 release ( Figure S6 C). Data are presented as mean ± SEM (A, B, C (-SEM only), D, E, F).

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Binding Assay, Concentration Assay, Transfection

Journal: iScience

Article Title: Perturbation of EPHA2 and EFNA1 trans binding amplifies inflammatory response in airway epithelial cells

doi: 10.1016/j.isci.2025.111872

Figure Lengend Snippet:

Article Snippet: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb , Cell Signaling Technology , Cat# 6347; RRID: AB_11220420.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, DNA Sequencing, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a FGF4 is a key signal that promotes differentiation of pluripotent mESCs. b Workflow for phosphoproteomic analysis of FGF4 signalling in Fgf4 −/− mESCs. Volcano plot showing significantly modified phosphosites after stimulation of Fgf4 −/− mESCs with FGF4 for 5 min ( c ) and 20 min ( d ). Phosphosites on known FGF4 pathway components are highlighted. e Protein kinase phosphopeptides that are significantly upregulated (>2-fold) on at least one time point (5 or 20 min) compared with control. Data are presented as mean ± SD ( n = 3). f Fgf4 −/− mESCs were stimulated with FGF4 for the indicated time, and EPHA2 pS898 and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Donor vectors containing Epha2 cDNA for exons 16 and 17, either WT or 5A, followed by an IRES2 and EGFP, were cloned into pMA (Addgene; MRC Reagents & Services DU60674, DU60744) and transfected in conjunction with the mentioned sgRNA-expressing vectors using Lipofectamine LTX (Life Technologies).

Techniques: Modification, Control, Western Blot

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Average protein copy number per cell determined for receptor kinases in mESCs, using quantitative whole-cell proteomics. Data are presented as mean ± SD ( n = 3). b Workflow for quantification of EPH–EFN interactions in mESCs by EFN ligand affinity purification mass spectrometry. c Proof-of-principle identification of EPH–EFN interactions by EFN ligand affinity purification. EPHA2 levels were determined by immunoblotting. d Coomassie staining of EFNA1/B1 affinity purification from mESCs. EFNA1, EFNB1 and EPHA2 proteins are indicated. (*) = non-specific band. e Mass-spectrometry analysis of 75–130-kDa region of the Coomassie stained EFNA1/EFNB1 affinity purification shown in ( d ). Total spectral counts recovered for each EPH receptor family member are indicated. f EFNA1/B1 affinity purification from intact Epha2 +/+ and pooled Epha2 −/− mESCs. Phosphotyrosine (pTyr), EPHA2 and ERK1/2 levels were determined by immunoblotting. Note that the pTyr signal is specific for EPHA2, and is not detected in the absence of EFN ligand. g EPHA2 was immunoprecipitated from Epha2 +/+ and Epha2 −/− mESCs, and pTyr, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting. h Epha2 +/+ mESCs were stimulated with EFNA1-expressing Epha2 −/− mESCs for 15 or 40 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Donor vectors containing Epha2 cDNA for exons 16 and 17, either WT or 5A, followed by an IRES2 and EGFP, were cloned into pMA (Addgene; MRC Reagents & Services DU60674, DU60744) and transfected in conjunction with the mentioned sgRNA-expressing vectors using Lipofectamine LTX (Life Technologies).

Techniques: Affinity Purification, Mass Spectrometry, Western Blot, Staining, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs (clone C4) stably expressing EPHA2 were cultured in the absence of LIF for 48 h. EPHA2, KLF4, DNMT3B, NANOG and OCT4 levels were determined by immunoblotting. b Epha2 +/+ or Epha2 −/− (clone C4) mESCs were maintained in 2i or differentiated in N2B27 media for 72 or 96 h, respectively, whereupon 10% of cells were replated in 2i. Total alkaline phosphatase staining is represented relative to Epha2 +/+ mESCs. The total number of alkaline phosphatase-positive colonies for Epha2 +/+ and Epha2 −/− mESCs is shown, and also represented relative to Epha2 +/+ mESCs. Data show mean ± SEM ( n = 3); statistical significance was determined using unpaired two-sided Student’s t test comparing Epha2 −/− with the Epha2 +/+ control (**** P < 0.0001, ** P = 0.0016). c Epha2 +/+ or Epha2 −/− (clone C4) mESCs stably expressing EFNA1, along with the respective parental controls, were grown in LIF/FBS, and KLF4, NANOG, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting, or EPHA2 immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. d Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs stably expressing EPHA2 were differentiated as embryoid bodies for 10 days, and the levels of Fgf5 , Brachyury , Mixl and Cer1 mRNA determined by qRT-PCR. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. The results shown are for technical replicates from two independent experiments, including three Epha2 −/− clones ( n = 3); statistical significance at day 4 was determined using unpaired two-sided Student’s t test comparing each group with the Epha2 +/+ control (ns = not significant, * P = 0.0252, ** P = 0.0045, *** P < 0.0001). e Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. ppERK1/2, total ERK1/2, STAT3 pY705 and total STAT3 levels were determined by immunoblotting. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. ppERK1/2 signal was quantified; data show mean ± SD ( n = 3); statistical significance was determined using one-sample two-sided t test comparing each group with control, theoretical mean = 1 (ns = not significant, 5 min; * P = 0.0467, 30 min; *** P = 0.0005, 45 min; ** P = 0.0014, 60 min; ** P = 0.0018). f Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. SHP2 was immunoprecipitated, and SHP2 and EPHA2 levels detected by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Donor vectors containing Epha2 cDNA for exons 16 and 17, either WT or 5A, followed by an IRES2 and EGFP, were cloned into pMA (Addgene; MRC Reagents & Services DU60674, DU60744) and transfected in conjunction with the mentioned sgRNA-expressing vectors using Lipofectamine LTX (Life Technologies).

Techniques: Stable Transfection, Expressing, Cell Culture, Western Blot, Staining, Control, Immunoprecipitation, Quantitative RT-PCR, Whisker Assay, Clone Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Diagram of potential phosphorylation sites within the EPHA2 S898 motif. Mass spectrometry analysis detects phosphorylation of at least three sites in EPHA2 immunoprecipitated from FGF4-stimulated Fgf4 −/− mESCs (see Supplementary Table ). b Fgf4 −/− mESCs were treated with 10 μM of the indicated inhibitors for 1 h, and stimulated with FGF4 for 10 min. EPHA2 pS898, EPHA2 and AKT pS473 levels were determined by immunoblotting. c Epha2 −/− mESCs were transfected with either wild type or 5E EPHA2 constructs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting and quantified. Data show mean ± SD ( n = 4). d EPHA2 was immunoprecipitated from EPHA2 WT knock-in (KI) or 5A KI cell lines and pTyr and EPHA2 levels determined by immunoblotting (upper panel). Relative pTyr/EPHA2 signal was quantified (lower panel). Data show mean ± SD ( n = 4). e Phase-contrast images of EPHA2 WT KI or 5A KI mESC lines; scale bar = 100 µM. f EPHA2 WT KI or 5A KI cell lines were cultured in LIF/FBS medium for 48 h, and KLF4, NANOG, DNMT3B, OCT4 ppERK1/2 and ERK1/2 levels determined by immunoblotting. g Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EPH receptor expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). h Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EFN ligand expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). Epha2 ( i ) and Efna1 ( j ) mRNA expression in 2i mESCs undergoing differentiation in N2B27 was determined by qRT-PCR analysis at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of two technical and three biological replicates ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Donor vectors containing Epha2 cDNA for exons 16 and 17, either WT or 5A, followed by an IRES2 and EGFP, were cloned into pMA (Addgene; MRC Reagents & Services DU60674, DU60744) and transfected in conjunction with the mentioned sgRNA-expressing vectors using Lipofectamine LTX (Life Technologies).

Techniques: Phospho-proteomics, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Knock-In, Cell Culture, Expressing, Quantitative RT-PCR, Whisker Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ mESCs were transfected with the indicated siRNAs, and EPHA2, OCT4 and SOX2 levels determined by immunoblotting. A non-specific band was used as a loading control. b Epha2 and Oct4 mRNA expression was determined by qRT-PCR following transfection of Epha2 +/+ mESCs with control or OCT4 siRNA. Data show mean ± SEM of four technical replicates (n = 4). c Pluripotency transcription factor-binding sites in Epha2 gene regulatory regions were extracted from CODEX mESC ChIP-SEQ data ( http://codex.stemcells.cam.ac.uk ). d Epha2 +/+ mESCs were transfected with the indicated siRNAs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated using EPHA2 antibody, and pTyr and EPHA2 levels determined by immunoblotting. e EFNA1/B1 affinity purification from control, dasatinib-treated or siOct4-transfected mESCs. pTyr, EPHA2, OCT4 and ERK1/2 levels were determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Donor vectors containing Epha2 cDNA for exons 16 and 17, either WT or 5A, followed by an IRES2 and EGFP, were cloned into pMA (Addgene; MRC Reagents & Services DU60674, DU60744) and transfected in conjunction with the mentioned sgRNA-expressing vectors using Lipofectamine LTX (Life Technologies).

Techniques: Transfection, Western Blot, Control, Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Immunoprecipitation, Affinity Purification

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: Expression (RPKM) of EPH ( a , b ) or EFN ( c , d ) mRNA in the inner cell mass (ICM) or trophectoderm (TE) of 64-cell mouse embryos ( a , c ) or 5-day human embryos ( b , d ). a , c n = 33 biologically independent cells for ICM, and n = 28 biologically independent cells for TE. b , d n = 73 biologically independent cells for ICM, and n = 142 biologically independent cells for TE. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. e EPHA2 regulation and function in mESCs. In the pluripotent state, OCT4 and other pluripotency factors promote EPHA2 receptor expression, enabling activation by EFNA ligands to support pluripotency by restraining ERK1/2. During differentiation, FGF4 drives ERK1/2–RSK activity to phosphorylate and inhibit EPHA2, whilst ERK1/2 suppresses an OCT4–EPHA2 transcriptional module to disable EPHA2 receptor expression. Source data are provided as a Source Data file.

Article Snippet: Donor vectors containing Epha2 cDNA for exons 16 and 17, either WT or 5A, followed by an IRES2 and EGFP, were cloned into pMA (Addgene; MRC Reagents & Services DU60674, DU60744) and transfected in conjunction with the mentioned sgRNA-expressing vectors using Lipofectamine LTX (Life Technologies).

Techniques: Expressing, Whisker Assay, Activation Assay, Activity Assay

Journal: PLoS ONE

Article Title: Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

doi: 10.1371/journal.pone.0036564

Figure Lengend Snippet: ( A ) Schematic diagram showing the domains of EPHA2 receptor and the locations of four SAM domain mutations found in human cataracts (c.2819C>T; c.2915_2916delTG; c.2826-9G>A; and c.2842G>T) in the EPHA2 gene. FN-III: fibronectin type-III domain; TM: transmembrane domain; Kinase: protein tyrosine kinase domain; SAM: sterile-α-motif domain; P: PDZ-binding motif. The SAM domain comprises 5 α-hecices (H1–5). ( B ) Reduction of mutant EPHA2 protein levels in transfected cells expressing EPHA2 mutants. Protein levels of EPHA2 mutants are decreased in both HEK293T and αTN4-1 cells. The blot was reprobed with anti-α-tubulin as a loading control. The graphs represent the quantification of relative band intensity of EphA2 as connected by the levels of α-tubulin from three independent experiments. Total EphA2 protein band intensity was determined using ImageJ software. Mean values are presented with S.D as indicated. Statistical differences between multiple groups were analyzed using one-way analysis of variance (ANOVA). ***, P <0.001; **, P <0.01; *, P <0.05; and ns, not significant. Values of P <0.05 were considered to be statistically significant. ( C, D ) No difference between wild-type and mutant EPHA2 genes in transcription levels. ( C ) Semi-quantitative RT-PCR and ( D ) Real-time PCR for wild-type and mutant EPHA2 genes were performed using total RNA, isolated from transfected HEK293T cells. GAPDH transcript levels are used as controls. The graphs represent the quantification of western blots from three independent experiments.

Article Snippet: HEK293T cells at 80% confluence were co-transfected with 2 μg of EPHA2 plasmid DNA and 1 μg of HA-tagged ubiquitin (HA-Ub) using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Sterility, Binding Assay, Mutagenesis, Transfection, Expressing, Control, Software, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation, Western Blot

Journal: PLoS ONE

Article Title: Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

doi: 10.1371/journal.pone.0036564

Figure Lengend Snippet: EphA2 knockout MEF cells expressing wild-type or mutant EPHA2 were incubated with clustered ephrin-A5-Fc before fixation and treated with anti-Fc antibodies (red). After washing with PBS, cells were counterstained with anti-EphA2 antibody (green). Images were captured using a Nikon Eclipse C1 confocal microscope. Scale bar, 50 µm.

Article Snippet: HEK293T cells at 80% confluence were co-transfected with 2 μg of EPHA2 plasmid DNA and 1 μg of HA-tagged ubiquitin (HA-Ub) using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Knock-Out, Expressing, Mutagenesis, Incubation, Microscopy

Journal: PLoS ONE

Article Title: Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

doi: 10.1371/journal.pone.0036564

Figure Lengend Snippet: ( A ) Mutant EPHA2 proteins have a reduced half-life. HEK293T cells were treated for indicated time with the protein biosynthesis inhibitor CHX (50 µg/mL) or the proteasome inhibitor MG132 (10 µM). Cell lysates were immunoblotted with anti-EphA2 antibody. Lysates were resolved by SDS-PAGE and western blot analysis was performed using indicated antibodies as described in the . The blot was reprobed with anti-α-tubulin as a loading control. ( B ) Quantification of EphA2 protein levels over time. Mean values are presented with S.D as indicated. ( C ) EPHA2 mutants have increased ubiquitination. Cells transfected with EPHA2 and HA-tagged ubiquitin were treated with 10 µM MG132 for 6 hours, and were then lysed. Immunoprecipitated EphA2 was further analyzed with western blotting using anti-HA antibodies to detect ubiquitinated EphA2 as described in the . The smear band is characteristic ubiquitin immunoreactivity. The amount of total EphA2 is shown as a loading control.

Article Snippet: HEK293T cells at 80% confluence were co-transfected with 2 μg of EPHA2 plasmid DNA and 1 μg of HA-tagged ubiquitin (HA-Ub) using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Mutagenesis, SDS Page, Western Blot, Control, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

doi: 10.1371/journal.pone.0036564

Figure Lengend Snippet: ( A, B ) Ephrin-A5 ligand stimulates EPHA2 phosphorylation. HEK293T ( A ) and αTN4-1 ( B ) cells were grown to confluence and growth factor-starved for 24 hours. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media and cell lysates were immunoblotted with indicated antibodies. Western blot analysis was performed as described in the . The blot was reprobed with anti-α-tubulin as a loading control. ( C ) The ratios of levels of phospho-EphA2 to total EphA2 are similar between the wild-type and mutant EPHA2 proteins. The graphs show total band intensity of anti-phospho-EphA2 immunoblot to total EphA2 and represent the average of three independent experiments. Quantification of phospho-EphA2 protein/total EphA2 protein levels was performed using ImageJ software. Mean values are presented with S.D as indicated. Statistical differences between multiple groups were analyzed using one-way analysis of variance (ANOVA). Values of P <0.05 were considered to be statistically significant. ns: No statistically significant difference between the two groups.

Article Snippet: HEK293T cells at 80% confluence were co-transfected with 2 μg of EPHA2 plasmid DNA and 1 μg of HA-tagged ubiquitin (HA-Ub) using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Mutagenesis, Software

Journal: PLoS ONE

Article Title: Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

doi: 10.1371/journal.pone.0036564

Figure Lengend Snippet: ( A ) Mutant EPHA2 genes fail to promote αTN4-1 cell migration. αTN4-1 cells were grown to confluency and serum-starved for 24 hours. A scratch wound was made with a micropipette tip and the edge of cells was marked. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media, and cells were allowed to migrate toward the center of the wound and photographed at the indicated times (representative figure of three independent experiments). The position of the initial scratch is indicated by dotted lines. Scale bar, 500 µm. ( B ) Quantification of the effects of EPHA2 genes on αTN4-1 cell migration. The graphs represent the measurement of migration distance from three independent experiments. Mean values are presented with S.D as indicated. Statistical differences were analyzed using one-way analysis of variance (ANOVA) or calculated by a two-tailed student t-test. Black asterisks , comparison between time 0 and 24 hours and time 0 and 48 hours; Blue asterisks , comparison between the mock groups and the listed wild-type or mutant EPHA2 genes at 24 or 48 hours; Red asterisks , comparison between untreated and treated conditions at 24 or 48 hours. ***, P <0.001; **, P <0.01; *, P <0.05; and ns, not significant. Values of P <0.05 were considered to be statistically significant.

Article Snippet: HEK293T cells at 80% confluence were co-transfected with 2 μg of EPHA2 plasmid DNA and 1 μg of HA-tagged ubiquitin (HA-Ub) using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Mutagenesis, Migration, Two Tailed Test, Comparison

Journal: PLoS ONE

Article Title: Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

doi: 10.1371/journal.pone.0036564

Figure Lengend Snippet: ( A, B ) Mutant EPHA2 proteins exhibit reduced activation of Akt and Erk by ephrin-A5. HEK293T cells were grown to confluence and serum-starved for 24 hours. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media and cell lysates were immunoblotted with anti-phospho-Akt (Ser473) or anti-phospho-Erk (1/2), and then reprobed with anti-α-tubulin as a loading control. ( C ) Inactivation of EphA2 gene leads to reduction of Akt activity in mouse lenses. Each lens was prepared from 22 days old mice and extracted with lysis buffer. Total lens proteins were resolved by SDS-PAGE and western blot analysis was performed using indicated antibodies as described in the . The blot was reprobed with anti-α-tubulin as a loading control.

Article Snippet: HEK293T cells at 80% confluence were co-transfected with 2 μg of EPHA2 plasmid DNA and 1 μg of HA-tagged ubiquitin (HA-Ub) using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Mutagenesis, Activation Assay, Control, Activity Assay, Lysis, SDS Page, Western Blot