Anti-Eph receptor A2/EPHA2 Antibody Picoband®

Western blot analysis of Eph receptor A2 using anti-Eph receptor A2 antibody (A00578).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human Hela cell lysate,
Lane 2: human U-87MG cell lysate,
Lane 3: human SHG-44 cell lysate,
Lane 4: human COLO-320 cell lysate,
Lane 5: human SK-OV-3 cell lysate,
Lane 6: human A549 cell lysate,
Lane 7: mouse HEPA1-6 cell lysate.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Eph receptor A2 antigen affinity purified polyclonal antibody (Catalog # A00578) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Eph receptor A2 at approximately 125KD. The expected band size for Eph receptor A2 is at 108KD.

Analysis of EPHA2 expression (A) High EPHA2 were associated with shorter relapse-free survival (RFS) in TNBC (Logrank p = 0.037) as demonstrated by Kaplan-Meier analysis. (B) According to the results of UCSC Xena in different BRCA subtypes, the expression level of EPHA2 was higher in TNBC than that in Luminal A ( p = 0.034), Luminal B ( p = 0.019) and normal-like ( p = 0.031) (Mann–Whitney test). (C) Relative protein expression of EPHA2 in different BRCA cell lines derived from metastatic tumors and primary tumors using DepMap portal. (D) The confirmation of relative protein expression of EPHA2 in different BRCA cell lines using western blot. *p < 0.05, **p < 0.01, ****p < 0.0001.
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Spatial distribution of cell types expressing EPHA2. (A) UMAP plots of stromal cells. (B) UMAP plots of epithelial cells. (C) EPHA2 expression distribution in stromal cells by subtype and celltype_subset. (D) EPHA2 expression distribution in epithelial cells by celltype_subset and subtype. (E) EPHA2 expression in different stromal cells. (F) EPHA2 expression in different epithelial cells. All these were analyzed using Single Cell Portal ( ). * p < 0.05, ns indicates no significant difference (Mann–Whitney test).
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EPHA2 knockdown affected MDA-MB-231 proliferation and invasion. (A) The verification of the knockdown efficiency of three EPHA2 shRNAs. (B) The transduction efficiency of EPHA2 shRNA was confirmed using Fluorescence microscopy. (C) Relative proliferation of MDA-MB-231 cells was decreased in EPHA2 shRNA group, compared to the control shRNA group ( p = 0.0007, t -test). (D) Knockdown of EPHA2 inhibited invasion of MDA-MB-231 as assessed by crystal violet staining ( p = 0.0003, t -test). Bars represent the mean ± SD. ***p < 0.001.
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EPHA2 knockdown induced MDA-MB-231 pyroptosis. (A) Volcano plot demonstrating an overview of the differential expression of all genes. (B) Enrichment ratio and enriched function of these DEGs was analyzed using KOBAS online server. C1-C5 represent different clusters. (C) STRING protein-protein interaction network. Proteins are represented as nodes while interactions appear as edges. Relative NLRP3 expression (D) and IL1β expression (E) in EPHA2 shRNA groups were all lower than in control shRNA groups ( p = 0.0006 and p = 0.0001, respectively, t -test). (F) The electron microscopy images of control shRNA and EPHA2 shRNA, red arrows indicating the apoptotic bodies for pyroptotic morphology. Bars represent the mean ± SD.
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EPHA2 knockdown induced pyroptosis pathway activation and inhibits PI3K/AKT/mTOR signal pathway activation. (A) Western blot analysis of NLRP3, GSDMD, caspase-1, AKT, p-AKT, PI3K, p-PI3K, mTOR and p-mTOR expression. * p <0.05, ** p < 0.01, *** p < 0.001. Statistical analysis of the two groups were performed using two-tailed Student’s t test. (B) Analysis of pathways and co-expression data for pyroptosis and genes related to the PI3K/AKT/mTOR signaling pathway using GeneMANIA. (C) Correlation of EPHA2 and IL18 expression in epithelial cells by subtype was analyzed based on Single Cell Portal. (D) The concentration of IL18 in cell culture supernatant of shEPHA2–1 was lower than in shControl RNA group ( p =0.0174, t -test). (E) Correlation of EPHA2 and IL1β expression in epithelial cells by celltype_subset was analyzed based on Single Cell Portal. (F) The concentration of IL1β in cell culture supernatant of shEPHA2–1 was lower than in shControl RNA group ( p =0.0259, t -test). Bars represent the mean ± SD.
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AKT inhibition induced MDA-MB-231 pyroptosis and decrease SLC7A11 expression. (A) Western blot analysis of NLRP3, GSDMD, caspase-1, AKT, p-AKT, PI3K, p-PI3K, mTOR and p-mTOR expression in MDA-MB-231 cells treated with AKT inhibitor or control buffer. * p <0.05, ** p < 0.01. Statistical analysis of the two groups were performed using two-tailed Student’s t test. The concentration of IL1β (B) and IL18 (C) in cell culture supernatant were all significantly elevated in the MK-2206-treated group, compared to the control group ( p = 0.0134 and p = 0.0015, respectively, t -test). (D) Analysis of pathways and co-expression data of SLC7A11, EHPA2, pyroptosis related genes and AKT/PI3K/mTOR related genes. Relative mRNA expression levels ( p = 0.0002, t -test) (E) and protein expression levels (F) of ferroptosis-associated gene SLC7A11 were all decreased in shEPHA2–1 group. Bars represent the mean ± SD.
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Flow Cytometry analysis of A549 cells using anti-Eph receptor A2 antibody (A00578).
Overlay histogram showing A549 cells stained with A00578 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Eph receptor A2 Antibody (A00578,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of U20S cells using anti-Eph receptor A2 antibody (A00578).
Overlay histogram showing U20S cells stained with A00578 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Eph receptor A2 Antibody (A00578,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IF analysis of Eph receptor A2 using anti-Eph receptor A2 antibody (A00578).
Eph receptor A2 was detected in immunocytochemical section of PC-3 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-Eph receptor A2 Antibody (A00578) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.