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Epha2

Manufactured by Santa Cruz Biotechnology
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Sourced in United States, China

EphA2 is a lab equipment product offered by Santa Cruz Biotechnology. It is a protein that plays a role in cell-cell communication and signaling processes. The core function of EphA2 is to act as a receptor tyrosine kinase, which mediates various cellular processes through the binding of its ligands.

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Lab products found in correlation

β actin, by Merck Group (4 mentions) Ndrg1, by Abcam (2 mentions) Gapdh, by Merck Group (2 mentions) Pakt s473, by Cell Signaling Technology (2 mentions) Rab27a, by Abcam (2 mentions) Tsg101, by Santa Cruz Biotechnology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ndrg1, by Cell Signaling Technology (1 mentions) Foxo3, by Cell Signaling Technology (1 mentions) Igf 1r, by Cell Signaling Technology (1 mentions) Epha2 ps897, by Cell Signaling Technology (1 mentions) Endogenous peroxidase blocker, by OriGene (1 mentions) P epha2, by Cell Signaling Technology (1 mentions) Protease and phosphatase inhibitor, by Roche (1 mentions) Ab124715, by Abcam (1 mentions) Vimentin, by Abcam (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Mmp 2, by Santa Cruz Biotechnology (1 mentions) Zb 2301, by Santa Cruz Biotechnology (1 mentions) Ve cadherin, by Abcam (1 mentions)

Spelling variants of this product

Anti-EphA2 (10 citations) Rabbit anti-EphA2 (4 citations) EphA2 siRNA (2 citations) Anti-EphA2 (sc-924, (2 citations) EphA2 antibody (2 citations) Anti-EphA2 (clone C-3, (2 citations)

14 protocols using epha2

1

Protein Analysis Using SDS-PAGE and Immunoblotting

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SDS-PAGE and immunoblotting were carried out as previously described (10 (link)). Cells were lysed with either radioimmunoprecipitation assay lysis buffer as previously described (10 (link)) or 1% Triton X-100 lysis buffer as described (46 (link)). Antibodies recognizing ERRB3 Y1197, ERBB3, EPHA2 S897, NDRG1 T346, Akt S473, Akt T308, Akt, p70S6K T389, p70S6K, ribosomal S6 S240/244, S6, IGF1R Y1135/36/IR Y1150/51, IGF1R, FOXO1/3a T24/T32, and FOXO3 were from Cell Signaling. Other antibodies included EPHA2 (Santa Cruz), secreted form of NRG1 (R&D Systems), NDRG1 (Abcam), and GAPDH (EMD Millipore). Anti-NF2/merlin polyclonal antibody has been previously described (61 (link)).
Beauchamp R.L., Erdin S., Witt L., Jordan J.T., Plotkin S.R., Gusella J.F, & Ramesh V. (2020). mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition. The Journal of Biological Chemistry, 296, 100157.
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2

Immunohistochemical Detection of KLF5, EphA2, and EphA2 pS897

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Briefly, the samples were fixed with 4% formaldehyde for 48 h at room temperature and embedded in paraffin. Then, the paraffin-embedded tissue sections at 5-8 μm thickness were transferred onto glass slides. The slides were deparaffinized, rehydrated, and pressure cooker heated for 2-5 min in EDTA for antigen retrieval. Endogenous peroxidase activity was inactivated by adding an endogenous peroxidase blocker (OriGene, Beijing, China) for 15 min at room temperature. Slides were incubated overnight at 4°C with KLF5 (Proteintech, 66850-1-Ig), EphA2 (Santa Cruz, sc-398832), and EphA2 pS897 (Cell Signaling, #6347) antibodies. Next, the slides were washed three times with 1×PBS and incubated with secondary antibody (OriGene, Beijing, China) at room temperature for 20 min, DAB concentrate chromogenic solution (1:200 dilution of concentrated DAB chromogenic solution), counterstained with 0.5% hematoxylin, dehydrated with graded concentrations of ethanol for 3 min each (70%-95%-100%), and finally stained with dimethyl benzene. Immunostained slides were evaluated by light microscopy. IOD score: integrated optical density score.
Zhao P., Sun J., Huang X., Zhang X., Liu X., Liu R., Du G., Gan W., Yang C., Tang Y., Chen C, & Jiang D. (2023). Targeting the KLF5-EphA2 axis can restrain cancer stemness and overcome chemoresistance in basal-like breast cancer. International Journal of Biological Sciences, 19(6), 1861-1874.
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3

Protein Extraction from Surgical Specimens

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Protein lysates were prepared from fresh surgical VS and GAN specimens directly after receiving samples from the operating room. Briefly, on ice, total protein was extracted from tumor or nerve using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche Applied Sciences), as previously described6 (link). Protein lysates for cultured SCs and mouse tumors were prepared using the same lysis conditions as VS and GAN samples. The resulting lysates were isolated by centrifugation and stored at −80 °C. Protein lysates were resolved by SDS-PAGE as previously described16 (link). Commercial antibodies included pEPHA2(S897), c-KIT, pSRC/SFK(Y416), SRC, pAkt(S473), Akt, SGK1, pNDRG1(T346), pS6(S240/244), and S6 (Cell Signaling Technology); NDRG1 (Abcam); EPHA2 (Santa Cruz Biotechnology); GAPDH (EMD Millipore); and β-actin (Sigma Aldrich). NF2/merlin polyclonal C26 antibody has been previously described26 (link). Quantitation of immunoblotting was performed using ImageJ/Fiji software27 (link).
Sagers J.E., Beauchamp R.L., Zhang Y., Vasilijic S., Wu L., DeSouza P., Seist R., Zhou W., Xu L., Ramesh V, & Stankovic K.M. (2020). Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma. Scientific Reports, 10, 4211.
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4

Comprehensive Protein Expression Analysis

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HepG2, HepG2-BMP4, SMMC7721, and SMMC7721-shBMP4 cells were lysed in 10% SDS. Cell lysates were first subject to SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore, USA) and then blocked in non-fat milk. Primary antibodies including BMP4 (ab124715, Abcam, USA), E-cadherin (zs-78700, Zhongshan Chemical Co., China), Vimentin (2707-1, Epitomics, USA), EphA2 (sc-924, Santa Cruz Biotechnology, USA), VE-cadherin (ab33168, Abcam, USA), MMP2 (sc-13595, Santa Cruz Biotechnology, USA), OCT4 (sc-8629, Santa Cruz Biotechnology, USA) and SOX2 (GTX101507, GeneTex, USA) were added to the membranes at 4°C overnight. The next day, the secondary antibodies including goat anti-rabbit IgG-HRP (ZB-2301, Santa Cruz Biotechnology, USA), goat anti-mouse IgG-HRP (ZB-2305, Zhongshan Chemical Co., China) and rabbit anti-goat IgG-HRP (ZB-2306, Zhongshan Chemical Co., China) were added for 2 h at 37°C. The protein levels were assessed using the enhanced chemiluminescence method.
Li X., Sun B., Zhao X., An J., Zhang Y., Gu Q., Zhao N., Wang Y, & Liu F. (2020). Function of BMP4 in the Formation of Vasculogenic Mimicry in Hepatocellular Carcinoma. Journal of Cancer, 11(9), 2560-2571.
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5

Immunofluorescence Imaging of Cellular Biomarkers

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Cells were grown and treated on chamber slides. At specified times after treatment with each inhibitor, cover slips were rinsed and cells were fixed in 4% paraformaldehyde then permeabilized in methanol for 20 minutes. Cells were subsequently washed and blocked in PBS containing 2% bovine serum albumin for 1 hour. Primary antibodies against γH2AX (Cell Signaling Technology), VEGF (Santa Cruz Biotechnology), EphA2 (Santa Cruz Biotechnology), LC3 (Cell Signaling Technology, Inc.), or Caspase-3 (Santa Cruz Biotechnology) were applied to the cells and incubated overnight. A secondary FITC anti-rabbit antibody (Molecular Probes, Eugene, OR, USA) was applied and incubated for 1 hour. DAPI nuclear counterstain was applied at 1 μg/mL for 5 minutes. Slides were examined on an Axio Scope.A1 Imager fluorescence microscope (Carl Zeiss, Gottingen, Germany. Images were captured using an AxioCam MRc5 and acquisition software AxioVision v.4.4 (Carl Zeiss).
Koo T., Cho B.J., Kim D.H., Park J.M., Choi E.J., Kim H.H., Lee D.J, & Kim I.A. (2017). MicroRNA-200c increases radiosensitivity of human cancer cells with activated EGFR-associated signaling. Oncotarget, 8(39), 65457-65468.
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6

Exosomal Protein Analysis by Western Blot

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Western blotting was performed as described previously22 . In brief, whole-cell lysates or exosomal proteins were separated by SDS–PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% milk for 1 h at room temperature and then incubated with the corresponding primary antibodies overnight at 4 °C. The following antibodies were used: TSG101 (sc-136111, Santa Cruz, CA, USA), CD81 (sc-23962, Santa Cruz), Alix (92880, CST, MA, USA), ERK (4695, CST), p-ERK (4370, CST), Akt (9272, CST), p-Akt (4051, CST), STAT3 (12640, CST), p-STAT3 (9145, CST), EphA2 (6997, CST), EphA2 (398832, Santa Cruz), Rab27a (ab55667, Abcam, MA, USA), and β-actin (A1978, Sigma-Aldrich, MO, USA). After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals were visualized with the ECL kit. CD81, Alix, and TSG101 were used as exosomal markers. β-actin was used as a loading control.
Gao Z., Han X., Zhu Y., Zhang H., Tian R., Wang Z., Cui Y., Wang Z., Niu R, & Zhang F. (2021). Drug-resistant cancer cell-derived exosomal EphA2 promotes breast cancer metastasis via the EphA2-Ephrin A1 reverse signaling. Cell Death & Disease, 12(5), 414.
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7

Quantifying EphA2 Phosphorylation in U251 Cells

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U251 EphA2-overexpressing cells were plated in 6-well dishes at a density of 100,000 cells/well and grown for 24 hours prior to stimulation with appropriate compounds for 30 min. 0.1% DMSO was used as vehicle control. Following treatment, cells were washed and lysed in modified RIPA Buffer (20 mM Tris-HCl pH 7.4, 20 mM NaF, 150 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% DCA, 2 mM EDTA, 1% Triton X-100, 2 mM Na3VO4, and protease inhibitors, including 1 mM phenylmethylsulphonyl fluoride, and 2 μg/ml each of aprotinin and leupeptin) for 20 min, followed by immunoprecipitation. Samples were boiled 5 min and run on 4-12% Bis-Tris Plus gels (Thermo Fisher), followed by transfer to Immobilon-P PVDF membranes (Millipore, Billerica, MA). Membranes were blocked 1 hr at room temperature in 3% BSA in TBS containing 0.05% Tween-20 (TBS-T) followed by overnight incubation with pEphA/B (1:500) and EphA2 (Santa Cruz) (1:1000) primary antibodies. Membranes were washed in TBS-T and incubated with goat anti-rabbit-HRP (1:5000) secondary antibody 1 hr at room temperature, followed by washing and developing with Luminol Reagent (Santa Cruz). Band intensities were quantified using ImageJ (NIH) to generate the figure.
Orahoske C.M., Li Y., Petty A., Salem F.M., Hanna J., Zhang W., Su B, & Wang B. (2020). Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth. Bioorganic & medicinal chemistry, 28(18), 115656.
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8

Western Blot Analysis of EphA2 and AKT Signaling

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Cell lysates were prepared using total protein extract kit (Keygen, China) according to the supplied protocol. Protein concentrations were measured by Pierce BCA Protein Assay Kit (Thermo, USA), following the manufacturer's protocol. The same amounts of protein (30μg) were resolved on 10% SDS-PAGE (Keygen, China) and transferred to PVDF membranes (Pierce Biotechnology, Inc., Rockford, IL, USA). Membranes were blotted overnight at 4oC with primary antibody. Rabbit antibodies against EphA2 (1:500, Santa Cruze), AKT (1:1000, Cell Signaling Technology), p-AKTS473 (1:1000, Cell Signaling Technology), p-EphA2S897 (1:1000, Cell Signaling Technology), GAPDH (1:5000, Cwbiotech, China), and goat anti-rabbit IgG-HRP (1:5000, Good-Science) were used for detecting the specific bands.
Wang H., Lin H., Pan J., Mo C., Zhang F., Huang B., Wang Z., Chen X., Zhuang J., Wang D, & Qiu S. (2016). Vasculogenic Mimicry in Prostate Cancer: The Roles of EphA2 and PI3K. Journal of Cancer, 7(9), 1114-1124.
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9

Lipid-based nanocarrier for siRNA delivery

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Labrafac
lipophile WL 1349 and Dynasan 116
were donated by Gattefossé (Lyon, France). siRNA EphA1, EphA2,
EphB3, and Control siRNA-FITC were both provided by Santa Cruz Biotechnology
(Freiburg, Germany). Docetaxel and DOTAP, MTT dye, and dimethyl sulfoxide
(DMSO) were obtained from Sigma-Aldrich (Steinheim, Germany). Taxotere
(20 mg/mL) was purchased from a pharmacy. Ultrapure water was obtained
by using a Milli-Q system (Millipore, Bedford, MO). All of the chemicals
were of analytical grade.
Şenel B., Başaran E., Akyıl E., Güven U.M, & Büyükköroğlu G. (2024). Co-Delivery of siRNA and Docetaxel to Cancer Cells by NLC for Therapy. ACS Omega, 9(10), 11671-11685.
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10

Immunoblotting Protocol for EphA2 and YAP/TAZ

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The following antibodies (1:1000 except where indicated) were used against the corresponding proteins: EphA2 (Santa Cruz #sc-924), EphA2 (CST #6997), pEphA2-Tyr588 (CST #12677S), pEphA2-Ser897 (1:500, Abgent #AP3722a), EFNA1 (R&D Systems, #AF702), YAP/TAZ (CST #8418), pYAP-Ser397 (CST #13619), pYAP-Ser127 (CST #13008), pTAZ-Ser89 (1:500, CST #75275), MLC (CST #3672), pMLC2-Ser19 (CST #3671), LATS1 (CST #3477), pLATS1-Ser909 (CST #9157), ERBB2 (Neomarkers #MS-730-P0), β-actin (Santa Cruz #sc-47778), and β-tubulin (Sigma #T4026). IRDye 680LT goat anti-mouse (Licor #925-68020, 1:20,000), IRDye 800CW goat anti-rabbit (Licor #925-32211, 1:10,000), horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Promega #W401B, 1:5000), and HRP-conjugated goat anti-mouse (Promega #W402B, 1:5000) were used as secondary antibodies. For immunoblotting, pre-cleared lysates were electrophoresed by SDS-PAGE and transferred to nitrocellulose membranes, which were blocked for 1 hr in 5% nonfat dry milk or 5% bovine serum albumin (BSA). Membranes were incubated with primary antibodies overnight followed by incubation with secondary antibodies for 1 hr at room temperature and imaged using Licor Odyssey or enhance chemiluminescence (Clarity ECL kit, Bio-rad). Densitometry was performed using ImageJ software, and measured proteins were normalized using tubulin controls.
Edwards D.N., Ngwa V.M., Wang S., Shiuan E., Brantley-Sieders D., Kim L.C., Reynolds A.B, & Chen J. (2017). Regulation of cancer glutamine metabolism by EphA2 RTK-dependent activation of transcriptional co-activators YAP and TAZ. Science signaling, 10(508), eaan4667.
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