- 38-0400 - Invitrogen Antibodies EFNA5 antibody

Submitted references Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality.

Jukonen J, Moyano-Galceran L, Höpfner K, Pietilä EA, Lehtinen L, Huhtinen K, Gucciardo E, Hynninen J, Hietanen S, Grénman S, Ojala PM, Carpén O, Lehti K
Scientific reports 2021 Apr 23;11(1):8856

Regulatory roles of ephrinA5 and its novel signaling pathway in mouse primary granulosa cell apoptosis and proliferation.

Worku T, Wang K, Ayers D, Wu D, Ur Rehman Z, Zhou H, Yang L
Cell cycle (Georgetown, Tex.) 2018;17(7):892-902

Epha2 and Efna5 participate in lens cell pattern-formation.

Zhou Y, Shiels A
Differentiation; research in biological diversity 2018 Jul - Aug;102:1-9

EphB signaling regulates target innervation in the developing and deafferented auditory brainstem.

Nakamura PA, Hsieh CY, Cramer KS
Developmental neurobiology 2012 Sep;72(9):1243-55

Diverse roles of Eph/ephrin signaling in the mouse lens.

Cheng C, Gong X
PloS one 2011;6(11):e28147

Wnt6 expression in epidermis and epithelial tissues during Xenopus organogenesis.

Lavery DL, Davenport IR, Turnbull YD, Wheeler GN, Hoppler S
Developmental dynamics : an official publication of the American Association of Anatomists 2008 Mar;237(3):768-79

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ephrin-A5-transfected HEK293 cells using Rb anti-ephrin-A5 (Product # 38-0400). Image courtesy of Dr. Elena Pasquale, The Burnham Institute, La Jolla, CA.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Ephrin-A5 was performed using 90% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Ephrin-A5 Rabbit Polyclonal Antibody (Product # 38-0400) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Ephrin-A5 was done on SH SY5Y cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Ephrin-A5 Rabbit Polyclonal Antibody (38-0400, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10, 000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 E-cadherin distribution in WT, ephrin-A5(-/-) and EphA2(-/-) lens capsule flat mounts. Fluorescent images reveal normal staining signals of E-cadherin in WT and EphA2(-/-) anterior epithelial cells (A and C) but alterations in the ephrin-A5(-/-) anterior epithelium (B, indicated by arrowheads). Three-dimensional reconstructions of z-stack images labeled for E-cadherin (green) and DAPI (blue, nuclei) of lens epithelial cells from P21 WT, ephrin-A5(-/-) and EphA2(-/-) lens capsule flat mounts (D, E and F). There is a notable disruption of E-cadherin staining in ephrin-A5(-/-) anterior epithelial cells as compared to those in WT and EphA2(-/-) cells. Scale bar, 20 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Localization of ephrin-A5 and EphA2 in the lens. Double immunolabeling of ephrin-A5 (red) and EphA2 (green) with DAPI staining (blue, nuclei) of anterior lens epithelial and fiber cells from lens capsule flat mounts of P21 wild-type (WT) and ephrin-A5(-/-), EphA2(-/-) mice (A). Side views of z-stack reconstructions of anterior epithelial cells with underlying fiber cells of P21 WT, ephrin-A5(-/-), EphA2(-/-) lens capsule flat mounts reveal that ephrin-A5 proteins (red) show punctate signals mostly at the lateral and apical sides of lens epithelial cells (EC), and EphA2 proteins (green) show diffused signals in fiber cells (F) (B). Scale bar, 20 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Canonical EphA2-pY588 activation remains unaltered or is even increased after depletion of EFNA5 . ( A ) Heat map for ephrin ligands and Eph receptor mRNAs in selected HGSC cell lines. Data obtained from CCLE and illustrated per cell line. ( B , C ) Quantification of EFNA1 ( B ) and EFNA5 ( C ) mRNAs in OVCAR3 and OVCAR4 with silenced EFNA1 or EFNA5 compared to the controls. N = 3. siScr is set to one. ( D - G ) EphA2 (total and phosphorylated) in OVCAR3 and OVCAR4 after silencing of EFNA1 or EFNA5 ( D ) along with EphA2-pY588 ( E ), EphA2-pS897 ( F ), and EphA2 ( G ) quantifications. N = 3. siScr is set to one. Full-length blots are presented in Supplementary Fig. 5 . p values (Student''s t-test): *< 0.05; **< 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 EphrinA5 protein expression is heterogeneous and associates with disease malignancy. ( A ) EphrinA5 IHC of a diagnosis-stage, treatment-naive, primary HGSC tumor. Magnified images for different tumor areas. ( B ) Representative images of ephrinA5 IHC used for scoring the ovarian tumor TMAs. ( C ) Correlation of ephrinA5 protein expression with different epithelial OC subtypes and benign tumors ordered according to the weighted arithmetic mean of ephrinA5 score (descending order). t = tissue type, M = malignant, B = benign, * = mostly benign. Pearson chi-square test showed highest overall ephrinA5 expression in HGSC (p = 9.00 x 10 -6 ).

38-0400

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