phoshpho epha2 s897  (Cell Signaling Technology Inc)

Journal: Virulence

Article Title: Mannan is a context-dependent shield that modifies virulence in Nakaseomyces glabratus .

doi: 10.1080/21505594.2025.2491650

Figure Lengend Snippet: Figure 3. N. glabratus mnn10Δ mutants induce higher phosphorylation of OEC EphA2 compared to their respective wild-type parental strains. Western blotting for total and phosphorylated EphA2 (pEpha2). Human α-actin, loading control. Figure is representative of three independent experiments. OECs, oral epithelial cells, negative control. SC5314, C. albicans reference strain, positive control. WT, wild-type strain. mnn10Δ, null mutant strain.

Article Snippet: Antibodies for assessment of EphA2 phosphorylation Anti-EphA2 (D4A2) and anti-p-EphA2 (S897) rabbit monoclonal antibodies were purchased from Cell Signaling Technologies (CST Inc., MA, USA) (catalogue numbers 6997S and 6347S, respectively).

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

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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: HA (clone #16B12, Biolegend, 1:1000 dilution), Flag (clone #1E6, Fujifilm, 1:1000 dilution), EPHA2 (clone #C-3, Santa Cruz Biotechnology (SCB), 1:2000 dilution), EFNA1 (clone #A-5, SCB, 1:200 dilution), Na + /K + ATPase (clone #H-3, SCB, 1:10000 dilution), phospho-S897 EPHA2 (clone #D9A1, CST, 1:500 dilution), phospho-Y772 EPHA2 (#8244, CST, 1:500 dilution).

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