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Rabbit Recombinant Monoclonal Eph receptor B2 antibody. Suitable for mIHC, IHC-P, WB, IHC-Fr and reacts with Human, Mouse, Rat samples.

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Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (AB252935), expandable thumbnail
  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (AB252935), expandable thumbnail
  • Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (AB252935), expandable thumbnail
  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (AB252935), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (AB252935), expandable thumbnail

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
mIHCIHC-PIPFlow CytWBICC/IFIHC-Fr
Human
Tested
Tested
Not recommended
Not recommended
Tested
Not recommended
Expected
Mouse
Expected
Tested
Not recommended
Not recommended
Tested
Not recommended
Tested
Rat
Expected
Tested
Not recommended
Not recommended
Expected
Not recommended
Tested

Tested
Tested

Species

Human

Dilution info

1/1000

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/1000

Notes

Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Species

Rat

Dilution info

1/1000

Notes

Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Species

Human

Dilution info

1/1000

Notes

Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Not recommended
Not recommended

Species

Mouse, Rat, Human

Dilution info

-

Notes

-

Not recommended
Not recommended

Species

Mouse, Rat, Human

Dilution info

-

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/1000 - 1/5000

Notes

-

Species

Human

Dilution info

1/1000 - 1/5000

Notes

-

Expected
Expected

Species

Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Not recommended
Not recommended

Species

Mouse, Rat, Human

Dilution info

-

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/250

Notes

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

Species

Rat

Dilution info

1/250

Notes

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

Expected
Expected

Species

Human

Dilution info

Use at an assay dependent concentration.

Notes

-

Associated Products

Select an associated product type

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Target data

Function

Receptor tyrosine kinase which binds promiscuously transmembrane ephrin-B family ligands residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. The signaling pathway downstream of the receptor is referred to as forward signaling while the signaling pathway downstream of the ephrin ligand is referred to as reverse signaling. Functions in axon guidance during development. Involved in the guidance of commissural axons, that form a major interhemispheric connection between the 2 temporal lobes of the cerebral cortex. Also involved in guidance of contralateral inner ear efferent growth cones at the midline and of retinal ganglion cell axons to the optic disk. In addition to axon guidance, also regulates dendritic spines development and maturation and stimulates the formation of excitatory synapses. Upon activation by EFNB1, abolishes the ARHGEF15-mediated negative regulation on excitatory synapse formation. Controls other aspects of development including angiogenesis, palate development and in inner ear development through regulation of endolymph production. Forward and reverse signaling through the EFNB2/EPHB2 complex regulate movement and adhesion of cells that tubularize the urethra and septate the cloaca. May function as a tumor suppressor. May be involved in the regulation of platelet activation and blood coagulation (PubMed:30213874).

Alternative names

DRT, EPHT3, EPTH3, ERK, HEK5, TYRO5, TYRO5, EPHB2, DRT, EPHT3, EPTH3, ERK, HEK5, Ephrin type-B receptor 2, Developmentally-regulated Eph-related tyrosine kinase, ELK-related tyrosine kinase, EPH tyrosine kinase 3, EPH-like kinase 5, Renal carcinoma antigen NY-REN-47, Tyrosine-protein kinase TYRO5, Tyrosine-protein kinase receptor EPH-3, EK5, hEK5

Recommended products

Rabbit Recombinant Monoclonal Eph receptor B2 antibody. Suitable for mIHC, IHC-P, WB, IHC-Fr and reacts with Human, Mouse, Rat samples.

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EPR22427-268

Purification technique

Affinity purification Protein A

Concentration
Loading...

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Eph receptor B2 also known as EphB2 is a member of the Eph receptor family which includes a number of receptor tyrosine kinases. It has a molecular weight of approximately 110 kDa. The B2 receptor is strongly expressed in various tissues including the brain spinal cord and developing embryos. These proteins include the Eph protein as well as other B2 proteins. The receptor plays a mechanical role in guiding cell positioning and tissue patterning by interacting with Ephrin ligands specifically ephrin-B2 and ephrin-B3.

Biological function summary

Eph receptor B2 functions in cell-cell communication and is involved in neuronal navigation and synaptic plasticity. It forms part of a complex that brings together ligand-receptor pairs on adjacent cells facilitating bidirectional signaling. The receptor's activation influences neuronal circuitry remodeling and helps in the differentiation of numerous cell types. The B2 protein acting as anti B2 modulates various cell signaling mechanisms essential for normal physiological functioning especially in the nervous system where it contributes to the intricate network of the eph protein interactions.

Pathways

These interactions place Eph receptor B2 in significant signaling pathways such as the MAPK/ERK and PI3K/Akt pathways. These play influential roles in cell proliferation survival and migration. The receptor interacts with numerous proteins including AB25AP through these pathways affecting developmental processes and mature cellular functions. The precise regulation of these pathways is necessary for maintaining homeostasis in complex biological systems especially within the context of the nervous system.

Associated diseases and disorders

Alterations in Eph receptor B2 expressions have been linked to conditions such as cancer and Alzheimer’s disease. In cancers overexpression of the B2 receptor can contribute to the abnormal growth and metastasis of tumor cells often through mechanisms involving interactions with other tumorigenic proteins like Eph receptor A2. In Alzheimer's disease changes in B2 receptor signaling are associated with disrupted synaptic plasticity leading to cognitive deficits highlighting the importance of the Eph B2 receptor and its interplay with related signaling proteins in disease pathways.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

15 product images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on the base of the crypts of human colon (PMID: 24151532) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

    Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    The molecular weight observed is consistent with what has been described in the literature (PMID: 21508957).

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure times: Lane 1: 70 seconds; Lanes 2- 4: 3 minutes.

    All lanes: Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/1000 dilution

    Lane 1: HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate at 20 µg

    Lane 2: Human brain tissue lysate at 20 µg

    Lane 3: Mouse brain tissue lysate at 20 µg

    Lane 4: Rat stomach tissue lysate at 20 µg

    Secondary

    Lanes 1, 3 and 4: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Lane 2: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution

    Predicted band size: 117 kDa

    Observed band size: 130 kDa

  • Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse colon tissue labeling Eph receptor B2 with ab252935 at 1/250 dilution (green), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on the membrane of epithelial cells, especially located in the basal crypt region (PMID: 19621336) is observed. Counterstained with DAPI (blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit used at a 1/1,000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Exposure time: Lane 1: 3 minutes; Lane 2: 3.25 seconds; Lane 3: 3 minutes

    Blocking and Diluting buffer and concentration: 5% NFDM/TBST

    All lanes: Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/5000 dilution

    Lane 1: His-tagged human EPHB1 recombinant protein, 10ng

    Lane 2: His-tagged human EPHB2 recombinant protein, 10 ng

    Lane 3: His-tagged human EPHB3 recombinant protein, 10 ng

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Predicted band size: 117 kDa

    Observed band size: 27 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on rat colon (PMID: 24151532) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

    Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat colon tissue labeling Eph receptor B2 with ab252935 at 1/250 dilution (green), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on the membrane of epithelial cells, especially located in the basal crypt region (PMID: 19621336) is observed. Counterstained with DAPI (blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit used at a 1/1,000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    The molecular weight observed is consistent with what has been described in the literature (PMID: 21508957).

    Exposure times: Lane 1: 3 minutes; Lane 2: 37 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Lane 1: Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/5000 dilution

    Lane 2: Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/1000 dilution

    Lane 1: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

    Lane 2: 4T1 (mouse mammary gland carcinoma epithelial cell), whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Predicted band size: 117 kDa

    Observed band size: 130 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on mouse colon (PMID: 24151532) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

    Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human bladder cancer (PMID: 24151532, 25148033) is observed. Counterstained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

    Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling Eph receptor B2 with ab252935 at 1/50 dilution (green), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. No staining in mouse liver. Negative control: liver (PMID: 19366806). Counterstained with DAPI (blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit used at a 1/1,000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    The upper band around 130 kDa is full length EPHB2 which is consistent to previous report (PMID: 21508957), while the lower band around 80 kDa could be non-specific band or cleaved fragment (PMID: 18713744). The expression on rat brain tissue is consistent with what has been described in the literature (PMID: 16840724).

    This blot was developed using a higher sensitivity ECL substrate.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    All lanes: Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/1000 dilution

    All lanes: Rat brain tissue lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Predicted band size: 117 kDa

    Observed band size: 130 kDa

    Exposure time: 15s

  • Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver tissue labeling Eph receptor B2 with ab252935 at 1/50 dilution (green), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. No staining in rat liver. Negative control: liver (PMID: 19366806). Counterstained with DAPI (blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit used at a 1/1,000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

  • Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections).

    Panel A: merged staining of anti-Muc2 (Anti-MUC2 antibody [EPR23479-47] ab272692, magenta; Opal™690), anti-Eph receptor B2 (ab252935, green; Opal™520) and anti-ErbB3 / HER3 (Anti-ErbB3 / HER3 antibody [SP71] - BSA and Azide free ab236220, red; Opal™570) on human colon. Panel B: anti-Muc2 stained on goblet cells. Panel C: anti-Eph receptor B2 stained on intestinal stem and progenitor cells. Panel D: anti-ErbB3 / HER3 stained on epithelial cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

    The section was incubated in three rounds of staining: in the order of Anti-MUC2 antibody [EPR23479-47] ab272692 at 1/2000 dilution (0.252 μg/ml), ab252935 at 1/1000 dilution  (0.527 μg/ml), and Anti-ErbB3 / HER3 antibody [SP71] - BSA and Azide free ab236220 at 1/3000 dilution (0.75 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

  • Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Fluorescence multiplex immunohistochemical analysis of the human jejunum (Formalin/PFA-fixed paraffin-embedded sections).

    Panel A: merged staining of anti-Muc2 (Anti-MUC2 antibody [EPR23479-47] ab272692, magenta; Opal™690), anti-Eph receptor B2 (ab252935, green; Opal™520) and anti-ErbB3 / HER3 (Anti-ErbB3 / HER3 antibody [SP71] - BSA and Azide free ab236220, red; Opal™570) on human jejunum. Panel B: anti-Muc2 stained on goblet cells. Panel C: anti-Eph receptor B2 stained on intestinal stem and progenitor cells. Panel D: anti-ErbB3 / HER3 stained on epithelial cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

    The section was incubated in three rounds of staining: in the order of Anti-MUC2 antibody [EPR23479-47] ab272692 at 1/2000 dilution (0.252 μg/ml), ab252935 at 1/1000 dilution  (0.527 μg/ml), and Anti-ErbB3 / HER3 antibody [SP71] - BSA and Azide free ab236220 at 1/3000 dilution (0.75 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

  • Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935), expandable thumbnail

    Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

    Western blot: Anti-EPHB2 antibody [EPR22427-268] (ab252935) staining at 1/3000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab252935 was shown to bind specifically to EPHB2. A band was observed at 130 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EPHB2 knockout cell line. To generate this image, wild-type and EPHB2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

    All lanes: Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/3000 dilution

    Lane 1: Wild-type HCT 116 cell lysate at 20 µg

    Lane 2: EPHB2 knockout HCT 116 cell lysate at 20 µg

    Lane 3: A549 cell lysate at 20 µg

    Lane 4: HepG2 cell lysate at 20 µg

    Secondary

    All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

    Performed under reducing conditions.

    Observed band size: 130 kDa

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