Name: Anti-VCAM1 antibody [EPR5047]
SKU: ab134047
Rating: 5 (16 reviews)

Anti-VCAM1 antibody [EPR5047] ab134047 is a rabbit monoclonal antibody that is used in VCAM1 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Validated on the Leica BOND™ RX automated IHC staining platform for IHC
- Antibody clone EPR5047 is the most widely used clone for VCAM1 on the market
- Specificity confirmed with Vcam1 knockout cell line validation

Images

Indirect ELISA - Anti-VCAM1 antibody [EPR5047] (AB134047), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% 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

	IHC-P	IP	I-ELISA	WB	ICC/IF	Flow Cyt (Intra)
Human	Tested	Tested	Expected	Tested	Tested	Tested
Mouse	Tested	Expected	Expected	Tested	Expected	Expected
Rat	Expected	Expected	Expected	Tested	Expected	Expected
Synthetic peptide	Not recommended	Not recommended	Tested	Not recommended	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500 - 1/1000	Notes Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.
Species Mouse	Dilution info 1/500 - 1/1000	Notes Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/40	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Human, Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/2000 - 1/10000	Notes Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples. Please see images below for recommended treatment conditions and positive controls.
Species Rat	Dilution info 1/2000 - 1/10000	Notes Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples. Please see images below for recommended treatment conditions and positive controls.
Species Human	Dilution info 1/2000 - 1/10000	Notes Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples. Please see images below for recommended treatment conditions and positive controls.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/250	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/40	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Associated Products

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Target data

See full target information VCAM1

Function

Cell adhesion glycoprotein predominantly expressed on the surface of endothelial cells that plays an important role in immune surveillance and inflammation (PubMed:31310649). Acts as a major regulator of leukocyte adhesion to the endothelium through interaction with different types of integrins (PubMed:10209034). During inflammatory responses, binds ligands on the surface of activated endothelial cells to initiate the activation of calcium channels and the plasma membrane-associated small GTPase RAC1 leading to leukocyte transendothelial migration (PubMed:22970700). Serves also as a quality-control checkpoint for entry into bone marrow by providing a 'don't-eat-me' stamping in the context of major histocompatibility complex (MHC) class-I presentation (PubMed:35210567).

Alternative names

CD106, Vascular cell adhesion protein 1, V-CAM 1, VCAM-1, INCAM-100, VCAM1

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR5047
Purification technique: Affinity purification Protein A
Concentration

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: Stable for 12 months at -20°C

Notes

Anti-VCAM1 antibody [EPR5047] (ab134047) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), I-ELISA, ICC/IF, IHC-P, IP and WB.

Anti-VCAM1 antibody [EPR5047] (ab134047) was first used in a scientific publication in 2013 and has been cited over 309 times in peer reviewed journals. It's performance in Western Blot and IHC in mouse and human samples is trusted by the scientific community.

Abcam's high quality manufacturing and validation processes ensure Anti-VCAM1 antibody [EPR5047] (ab134047) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

The specificity of Anti-VCAM1 antibody [EPR5047] (ab134047) has been confirmed by Western Blot testing in VCAM1 knockout A549 cells (Human VCAM1 knockout A549 cell line ab273758).

Anti-VCAM1 antibody [EPR5047] (ab134047) has 16 independent reviews from customers.

Anti-VCAM1 antibody [EPR5047] (ab134047) specifically detects VCAM1 (UniProt ID: P29533; Molecular weight: 79kDa) and is sold in 100 µL and 1 mL selling sizes.

Conjugation-ready, carrier free format available for antibody clone EPR5047 - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free ab215380.

Antibody clone EPR5047 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor^® 647, HRP, PE, FITC, APC (Alexa Fluor® 647 Anti-VCAM1 antibody [EPR5047] ab194319, HRP Anti-VCAM1 antibody [EPR5047] ab195540, PE Anti-VCAM1 antibody [EPR5047] ab223981, FITC Anti-VCAM1 antibody [EPR5047] ab223982, APC Anti-VCAM1 antibody [EPR5047] ab223983).

- Specificity confirmed with SYP knockout cell line validation

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

VCAM1 also known as vascular cell adhesion molecule 1 plays an important role in mediating adhesion and signal transduction. VCAM1 has an approximate molecular mass of 110 kDa. It is expressed on the surface of endothelial cells and can be upregulated by cytokines released during inflammation. The protein serves as a ligand for the integrin VLA-4 also known as α4β1 and contributes to the adhesion of leukocytes to endothelial cells.

Biological function summary

Adhesion molecules like VCAM1 participate in immune response by recruiting leukocytes to sites of inflammation. VCAM1 helps guide immune cells to directed locations where they perform defense activities. Although VCAM1 is not part of any known large protein complex its interaction with integrins facilitates the migration of immune cells across the endothelium and into tissue.

Pathways

VCAM1 contributes significantly to the leukocyte extravasation process in inflammatory pathways. It plays a role in both the cytokine-cytokine receptor interaction pathway and the NF-κB signaling pathway which are essential for immune response and cell signaling. Through these pathways it interacts with proteins like ICAM1 and various chemokines helping to coordinate the immune cell movement through vascular tissue barriers.

Associated diseases and disorders

Abnormal regulation of VCAM1 links to various cardiovascular diseases such as atherosclerosis and inflammatory diseases like rheumatoid arthritis. The protein levels may increase in these conditions promoting excessive immune cell recruitment and tissue damage. VCAM1 also interfaces with other proteins like ICAM1 in these pathological states which together contribute to the progression and severity of these diseases.

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.

18 product images

Indirect ELISA - Anti-VCAM1 antibody [EPR5047] (ab134047)
ELISA using ab134047 at varying antibody concentrations and antigen concentration at 250 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Lanes 1 - 6: Merged signal (red and green). Green - ab134047 observed at 105 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab134047 was shown to react with VCAM1 in treated wild-type A549 cells in Western blot with loss of signal observed in treated VCAM1 knockout cell line Human VCAM1 knockout A549 cell line ab273758 (knockout cell lysate Human VCAM1 knockout A549 cell lysate ab275504). Wild-type A549 and VCAM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab134047 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/2000 dilution
Lane 1: Wild-type A549 cell lysate at 30 µg
Lane 2: Wild-type A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg
Lane 2: Western blot - Human VCAM1 knockout A549 cell line (Human VCAM1 knockout A549 cell line ab273758)
Lane 3: VCAM1 knockout A549 cell lysate at 30 µg
Lane 4: VCAM1 knockout A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg
Lane 5: HUVEC cell lysate at 30 µg
Lane 6: HUVEC TNF-a treated (16 ng/mL, 16h) cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 105 kDa
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Rabbit monoclonal [EPR16891] to GAPDH (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) used as loading control.
Blocking/Diluting buffer: 5% NFDM/TBST.
Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/1000 dilution
Lane 1: HUVEC (Human umbilical vein endothelial cell) whole cell lysate at 20 µg
Lane 2: HUVEC (Human umbilical vein endothelial cell) treated with 10 ng/ml TNF-α for 16 h, whole cell lysate at 20 µg
Lane 3: bEnd.3 (Mouse brain endothelioma) whole cell lysate at 20 µg
Lane 4: bEnd.3 (Mouse brain endothelioma) treated with 10 μg/ml LPS for 24 h, 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 1/20000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa, 36 kDa
Exposure time: 2s
Immunoprecipitation - Anti-VCAM1 antibody [EPR5047] (ab134047)
VCAM1 was immunoprecipitated from Human fetal liver lysate with ab134047 at 1/110 dilution.
Western blot was performed from the immunoprecipitate using ab134047 at 1/1000 dilution.
VeriBlot for IP secondary antibody (Peroxidase conjugated),was used as secondary antibody at 1/1000 dilution.
Lane 1: Human fetal liver lysate.
Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 1 second.
All lanes: Immunoprecipitation - Anti-VCAM1 antibody [EPR5047] (ab134047)
Predicted band size: 81 kDa
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Rabbit monoclonal [EPR16891] to GAPDH (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) used as loading control.
Blocking/Diluting buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/1000 dilution
Lane 1: Hut-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysate at 20 µg
Lane 2: SK-OV-3 (Human ovarian cancer epithelial cell) whole cell lysate at 20 µg
Lane 3: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg
Lane 4: J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate at 20 µg
Lane 5: LADMAC (Mouse bone marrow monocyte macrophage) 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 1/20000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa, 36 kDa
Exposure time: 7s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] (ab134047)
Immunohistochemical staining of paraffin embedded human tonsil with purified ab134047 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
Immunocytochemistry/ Immunofluorescence - Anti-VCAM1 antibody [EPR5047] (ab134047)
Immunofluorescent staining of K562 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab134047 at a dilution of 1/250. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counter stained with DAPI. The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.
Immunocytochemistry/ Immunofluorescence - Anti-VCAM1 antibody [EPR5047] (ab134047)
ab134047 staining VCAM1 in HUVEC cells treated with TNF-α (Recombinant Human TNF alpha protein ab55237) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab134047 at 2μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 647) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed ab150119) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution
Lane 1: Mouse kidney at 20 µg
Lane 2: Rat spleen at 20 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] (ab134047)
IHC image of VCAM1 staining in Human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Blocking buffer: 5% NFDM/TBST.
Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/2000 dilution
All lanes: Rat kidney tissue lysate at 20 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Blocking buffer: 5% NFDM/TBST.
Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution
All lanes: NIH/3T3 cell lysate at 10 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Blocking buffer: 5% NFDM/TBST.
Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution
All lanes: Human fetal liver tissue lysate at 20 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
Blocking buffer: 5% NFDM/TBST.
Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution
All lanes: HuT-78 cell lysate at 10 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 81 kDa
Observed band size: 100 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] (ab134047)
IHC image of VCAM1 staining in Mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047)
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/1000 dilution
Lane 1: Human fetal liver tissue lysate at 10 µg
Lane 2: HuT 78 cell lysate at 10 µg
Lane 3: NIH/3T3 cell lysate at 10 µg
Lane 4: Mouse brain tissue lysate at 10 µg
Lane 5: Mouse kidney tissue lysate at 10 µg
Lane 6: Mouse spleen tissue lysate at 10 µg
Lane 7: Rat brain tissue lysate at 10 µg
Lane 8: Rat kidney tissue lysate at 10 µg
Lane 9: Rat spleen tissue lysate at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab6721) at 1/2000 dilution
Predicted band size: 81 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] (ab134047)
Immunohistochemical analysis of formalin-fixed paraffin-embedded human tonsil labelling VCAM1 with Anti-VCAM1 antibody [EPR5047] - BSA and Azide free ab271899 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). Anti-VCAM1 antibody [EPR5047] - BSA and Azide free ab271899 anti VCAM1 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
This data was developed using the same antibody clone in a different buffer formulation (Anti-VCAM1 antibody [EPR5047] - BSA and Azide free ab271899).
Flow Cytometry (Intracellular) - Anti-VCAM1 antibody [EPR5047] (ab134047)
Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling VCAM1 with purified ab134047 at 1/40 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.