biotinylated epha2  (Sino Biological)

Journal: PLOS Pathogens

Article Title: Monoclonal neutralizing antibodies elicited by infection with Kaposi sarcoma-associated herpesvirus reveal critical sites of vulnerability on gH/gL

doi: 10.1371/journal.ppat.1013772

Figure Lengend Snippet: (A) Plasmids encoding the gH ectodomain and gL were co-transfected into 293 cells and recombinant gH/gL was purified from the supernatant. Made with BioRender.com. McGuire, A. (2025). https://BioRender.com/ks0xtn4 . (B) Recombinant gH/gL was subjected to size exclusion chromatography on a BioRad ENrichSEC 650 10 x 300 column. Three fractions were collected as indicated. (C) An aliquot of the gH/gL preparation pre-SEC, as well as equal amounts of the three fractions collected from B were analyzed by reducing SDS-PAGE followed by Coomassie staining. (D) Coomassie stained reducing SDS-PAGE gel of recombinant gH/gL treated, or untreated with PNGaseF as indicated. (E) Binding of recombinant gH/gL or recombinant Epstein-Barr virus gp350 to recombinant ephrin receptor A2 (EphA2) was measured by biolayer interferometry.

Article Snippet: Biotinylated EphA2 (Sino Biological Cat: 13926-H27H-B) was diluted to 10 μg/ml and immobilized on streptavidin biosensors for 300s, and then immersed in KB buffer for 60 seconds.

Techniques: Transfection, Recombinant, Purification, Size-exclusion Chromatography, SDS Page, Staining, Binding Assay, Virus

Journal: PLOS Pathogens

Article Title: Monoclonal neutralizing antibodies elicited by infection with Kaposi sarcoma-associated herpesvirus reveal critical sites of vulnerability on gH/gL

doi: 10.1371/journal.ppat.1013772

Figure Lengend Snippet: (A) gH/gL was incubated with, or without the indicated mAbs and the binding to immobilized EphA2 was measured by biolayer interferometry. The dashed line represents 100% binding set to the gH/gL alone control. (B-C) MLKH1/gH/gL (B) and MLKH5/gH/gL (C) complexes were purified and visualized by negative stain EM (nsEM). A representative 2D class average (top) and 3D reconstruction (bottom) are shown for each complex. The ribbon structure of gH/gL (PDB entry 7CZF, gH in black, gL in yellow) with each Fab (modeled using Alphafold) was fitted into the nsEM maps. (D) The crystal structure of the EphA2/gH/gL complex (PDB entry 7CZE) is shown for comparison.

Article Snippet: Biotinylated EphA2 (Sino Biological Cat: 13926-H27H-B) was diluted to 10 μg/ml and immobilized on streptavidin biosensors for 300s, and then immersed in KB buffer for 60 seconds.

Techniques: Incubation, Binding Assay, Control, Purification, Staining, Comparison

Journal: Methods (San Diego, Calif.)

Article Title: Rapid and improved surface passivation method for Single-Molecule experiments ☆

doi: 10.1016/j.ymeth.2026.01.003

Figure Lengend Snippet: Representative TIRF images of immobilized EphA2-GFP DIBMALPs in control (A) and EA1-treated (C) conditions (≤5 nM). Examples of 1-, 2-, and ≥ 3 step (EA1-treated only) photobleaching traces of EphA2-GFP in the presence (D) and absence (B) of its ligand EA1. (E) Quantification of photobleaching steps of EphA2-GFP in DIBMALPs with and without ligand treatment. A total of 126 molecules were analyzed for control and 177 in the presence of EA1.

Article Snippet: DIBMALPs containing EphA2-GFP (≤5 nM) were immobilized by a biotinylated EphA2 antibody (Cell Signaling) and illuminated by a 465 nm laser and imaged by TIRF microscopy as described above and in [ ].

Techniques: Control

Journal: OncoImmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402x.2025.2518631

Figure Lengend Snippet: Figure 1. GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104– 503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: OncoImmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402x.2025.2518631

Figure Lengend Snippet: Figure 3. In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3+ cells (NT) or live single CD3+/EGFP+ cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70+/CD276+/EPHA2+) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t0 = 0 min, t1 = 140 min, t2 = 280 min, t3 = 420 min, t4 = 560 min. For a-CD276: t0 = 0 min, t1 = 220 min, t2 = 440 min, t3 = 660 min, t4 = 880 min. For a-CD70: t0 = 0 min, t1 = 350 min, t2 = 700 min, t3 = 1050 min, t4 = 1400 min. N = 2 biological replicates

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104– 503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: OncoImmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402x.2025.2518631

Figure Lengend Snippet: Figure 5. Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro. (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP−cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin- V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP−/Annexin-Vhigh tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104– 503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Sample Prep, Membrane, Microscopy, Imaging

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: Western blot analysis of EphA2 pS897 in HEK293T (A) , A375 (B) and A431 (C) cells. We show pS897 quantification (mean ± S.D) and representative blots. p -values from one-way ANOVA followed by Mann-Whitney U or t test. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) cAMP quantification in HEK293T cells transduced with Red Up cADDis cAMP biosensor ollowing treatment with MβCD. (B) Western blot analysis and quantification of PKA T197 phosphorylation in A375 cells following treatment with MβCD. (C-D) Western blot analysis and quantification of EphA2 S897 phosphorylation in A375 cells following treatment with forskolin and soproterenol, respectively. Data shown in panel B are normalized to the respective total PKA signal in Figure S15A. Data shown in panels C-D are normalized to the respective total EphA2 signal in Figure S15B-C. Quantitative comparisons between treatments were made with respect to normalized control conditions. Bar graphs show mean ± S.D., p -values in panels A-D are from an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

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

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) In the presence of normal levels of Chol (yellow oval), EphA2 (blue) can be found as a monomer and displays low Ser897 phosphorylation. We propose that in this state the kinase domain interacts with the SAM domain. (B) When Chol content is reduced, β-AR (purple) activity increases, promoting cAMP/PKA signaling that enhances Ser897 phosphorylation (red dot). This forces the kinase-SAM linker into an open conformation, which promotes higher-order oligomers independent from ligand stimulation.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Phospho-proteomics, Activity Assay