Phospho-ITGA4 (S1027) Antibody
Description
Introduction to Phospho-ITGA4 (S1027) Antibody
The Phospho-ITGA4 (S1027) Antibody is a highly specific research-grade reagent designed to detect phosphorylated Integrin alpha-4 (ITGA4) at serine residue 1027. ITGA4, also known as CD49d, is a key component of the integrin family, forming heterodimers with beta1 or beta7 subunits (VLA-4 and alpha4beta7 receptors, respectively). These receptors mediate cell adhesion, migration, and signaling in immune cells, endothelial cells, and smooth muscle cells .
The antibody is a polyclonal rabbit IgG, affinity-purified for specificity to the phosphorylated epitope. Its primary applications include immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) .
Key Specifications (Table 1):
| Parameter | Details |
|---|---|
| Host | Rabbit |
| Reactivity | Human, Mouse |
| Immunogen | Synthetic peptide (983-1032 aa) |
| Dilution Range | IHC: 1:100-300; IF: 1:200-1000 |
| Storage | -20°C, 1-year shelf life |
| Conjugation | Unconjugated |
Protein Structure
ITGA4 is a single-pass type I membrane protein with a large extracellular domain and a short cytoplasmic tail. Phosphorylation at Ser1027 inhibits binding to paxillin (PXN), a critical step in regulating integrin activation and cell adhesion .
Biological Roles
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Immune Cell Migration: ITGA4 facilitates lymphocyte homing to the gut (via alpha4beta7/MADCAM1 interaction) and adhesion to endothelial VCAM1 .
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Vascular Pathology: Upregulated in Kawasaki Disease (KD), promoting inflammatory cell recruitment and smooth muscle cell proliferation .
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Neuroinflammation: Targeted by Natalizumab (anti-ITGA4) in multiple sclerosis (MS) to inhibit immune cell infiltration into the CNS .
Disease Models
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Kawasaki Disease: ITGA4 phosphorylation correlates with vasculopathy severity, as shown in studies using immunohistochemistry on coronary artery tissues .
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Multiple Sclerosis: Natalizumab therapy highlights ITGA4’s role in immune cell trafficking, with the antibody aiding mechanistic studies .
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Cancer: ITGA4 expression is linked to tumor lymphangiogenesis and metastasis, with phosphorylation modulating fibronectin/VCAM1 binding .
Cellular Mechanisms
Product Specs
Target Background
- Identifies molecularly distinct subsets of articular cartilage enriched for osteochondral proteins. PMID: 30194383
- Suggests a novel association of ITGA4 +3061A/G polymorphism with Alzheimer's disease (AD) and its possible contribution to the disease pathology. PMID: 29769839
- Demonstrates that aggregation of human Wharton's jelly stem cells (hWJSCs) into spherules alters their expression of ITGA4 and ITGA5. PMID: 29411210
- Shows that human ADAM11, ADAM23, and ADAM29 selectively support integrin alpha4-dependent cell adhesion. PMID: 28913673
- Reports that recombinant human IL33 inhibits trophoblast invasion and adhesion, and decreases the expression of adhesion and invasion-associated molecules such as integrin alpha4beta1 and CD62L. PMID: 28765940
- Analyzes for the first time the expression of epithelial-mesenchymal transition marker proteins - periostin, integrin alpha4, fibronectin - in metastatic castration-resistant prostate cancer. PMID: 28836852
- Highlights the roles of ITGalpha4beta1 and TLR4 in inducing fibrotic gene expression in response to fibronectin's EDA domain. PMID: 28842322
- Demonstrates that ITGA4 and PLCG1 are regulated by miR-30b in coronary artery endothelial cells from coronary atherosclerosis clinical samples. PMID: 27464494
- Reports that low CD49D expression is associated with chronic lymphocytic leukemia compared to small lymphocytic lymphoma. PMID: 29153094
- Shows that alpha-Integrin expression and function modulate presentation of cell surface calreticulin. PMID: 27310876
- Demonstrates that AEbeta7 plays a crucial role in gut trafficking of inflammatory bowel disease (IBD) CD8(+) T cells and CD4(+) Th9 cells in vivo, and mainly retention might account for this effect. These findings suggest that blocking alphaEbeta7 in addition to alpha4beta7 could be particularly effective in intestinal disorders characterized by expansion of CD8(+) and Th9 cells, such as IBD. PMID: 27543429
- Reviews the allosteric activation of Integrin alphaVbeta3 and Integrin alpha4beta1 by sPLA2-IIA. PMID: 27864802
- Concludes that analyzing alpha4beta7 integrin expression on circulating lymphocytes is not a reliable test for gastrointestinal involvement in common variable immunodeficiency (CVID) patients. PMID: 28323147
- Finds that glycosylation of the V1V2 domain of HIV envelope protein determines its binding to alpha4ss7 integrin. PMID: 28577856
- Confirms CD49d as an independent negative overall survival (OS) prognosticator in chronic lymphocytic leukemia (CLL) even in comprehensive models including novel recurrent mutations. PMID: 27109509
- Reports a positive correlation between the expression of integrin alpha4 and VEGF-C in colon cancer samples. Integrin alpha4 and VEGF-C were significantly associated with clinicopathological parameters (lymph node metastasis, Duke's stage, and local lymph node metastasis). Patients with high integrin alpha4 or VEGF-C expression had significantly shorter overall survival and tumor-free survival time. PMID: 26917449
- Shows that macrophage tissue factor prothrombotic activity is regulated by integrin-alpha4/arf6 trafficking. PMID: 28495929
- Demonstrates that gut-homing alpha4beta7 CD4(+) T cells and their functional subsets were profoundly depleted during acute HIV-1 infection. PMID: 26277899
- Suggests that CD49d determination may be a useful tool to closely monitor Multiple Sclerosis activity in patients who interrupt Natalizumab. PMID: 27496090
- Reports that Rho kinase (ROCK) activity was increased by inhibiting integrin alpha4 with human umbilical vein endothelial cells (HUVECs) and hypoxia. PMID: 26448639
- Demonstrates that Mycobacterium tuberculosis ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to fibronectin through alpha4beta1 integrin. PMID: 25081983
- Reviews the multiple functional roles of VLA-4 in cancer and presents this integrin as a promising target worthy of attention in biomedical cancer research. PMID: 25564456
- Hypothesizes that the increased susceptibility of activated endocervical CD4+ T cells to HIV may be related to increased CCR5 expression by these cell subsets, but not due to direct interaction of integrins a4b7 or a4b1 with HIV envelope. PMID: 25872482
- Reports that knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4), ITGA5, and integrin beta 1 (ITGB1), while RhoA expression was significantly decreased. PMID: 26900962
- Shows that ITGA4 expression enhances metastasis in MYCN low neuroblastoma. PMID: 25973900
- Suggests that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using an integrin alphavbeta3 and VLA-4-targeted nanotherapy approach. PMID: 25824336
- Presents data suggesting a role for alpha4beta7 integrin in HIV infection influenced by both viral and host factors, including the sequence of the HIV gp120 alpha4beta7 binding motif, the cytokine milieu, and bacterial vaginosis in the genital tract. PMID: 26105197
- Reports that glioma cells induced the migration of bone marrow stromal cells (BMSCs) by promoting the expression of integrin a4. PMID: 26034878
- Shows that CD49d and CD26 have independent prognostic value and recommends their use as part of a routine panel for prognostic stratification of CLL. PMID: 26142332
- Reports that hypermethylation of ITGA4 promoter is increased in inflamed colon tissue. PMID: 25902909
- Demonstrates that regulatory CD4 T cells (Treg) cells in co-infected patients present phenotypic alterations and might have dysfunction marked by low expression of Foxp3 and increased expression of molecules not frequently seen on Treg cells, such as CD49d. PMID: 26329520
- Reports that high VLA-4 expression was independently associated with a high probability of complete remission in acute myeloid leukemia patients. PMID: 26155911
- Shows that integrin activation is absolutely required for osteopontin (OPN) binding to the alpha4 integrin, suggesting that OPN has very low affinity for the alpha4 integrin on human leukocytes under physiological conditions. PMID: 25446551
- Reports that alpha4beta7 serves as an attachment factor for some HIV-1 strains. PMID: 25527342
- Shows that selective inhibition of VLA-4 expression on B cells impedes central nervous system accumulation of B cells and reduces susceptibility to experimental autoimmune encephalomyelitis. PMID: 25712734
- Demonstrates that the carbon monoxide signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand. PMID: 25367365
- Reports that CXCR4 and CD49d are important modulators of prognosis in chronic lymphocytic leukemia patients. PMID: 25349153
- Demonstrates that proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site in integrin alphaVbeta3, integrin alphavbeta1, and integrin alpha4beta1. PMID: 25398877
- Highlights the role of LFA-1 and CR4 alpha chain phosphorylation in chemokine-induced cross-talk to VLA-4. PMID: 25278023
- Reports that HIV-1 virions and many gp120s lack detectable alpha4beta7 binding activity. PMID: 25008916
- Shows that lymphocyte trafficking into the central nervous system (CNS) under VLA-4 blockade can occur through alternative adhesion molecules, PSGL-1 and MCAM, with the latter representing an exclusive pathway for TH17 cells to migrate over the blood-brain barrier. PMID: 25135296
- Suggests that DNA methylation of integrin alpha4 may be a poor prognostic factor affecting undifferentiated histologic change of breast cancer. PMID: 24756760
- Reports that disruption of hydrophobic contacts induces the active conformation of integrin alpha 4 beta 7. PMID: 24802248
- Shows that pharmacological inhibition of the VLA-4/vascular adhesion molecule-1 axis in experimental stroke was ineffective. PMID: 24743435
- Indicates that CD47 in cis interactions regulate LFA-1 (integrin alphaLbeta2) and VLA-4 (integrin alpha4beta1) integrin affinity, and this process plays a substantial role in T-cell adhesion. PMID: 24006483
- Demonstrates that VCAM-1/VLA-4-dependent activation of NF-kappaB plays a role in mediating chemoresistance in leukemia cells. PMID: 24599548
- Shows that two conserved disulfide bonds located at integrin alpha4C589-C594 and beta7C494-C526 activated alpha4beta7. PMID: 23986478
- Reports that only rotavirus-specific CD4 T cells expressed intestinal homing receptors alpha4beta7 and CCR9. PMID: 24606696
- Reviews CD38 (CD38 antigen (p45) protein) and CD49d (alpha4 Integrin; very late antigen-4 alpha) as more than just markers of an aggressive chronic lymphocytic leukemia (CLL) cell type, highlighting their functional roles in the pathobiology of CLL. PMID: 24288111
- Suggests that expression of the VLA-4-stimulating factor sequence may help to predict melanoma prometastatic risk. PMID: 23938462
Q&A
Basic Research Questions
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What is the significance of ITGA4 Ser1027 phosphorylation in cellular function?
Phosphorylation of Integrin alpha-4 (ITGA4) at serine 1027 plays a critical regulatory role in cell adhesion and migration. When ITGA4 becomes phosphorylated at Ser1027, it specifically inhibits paxillin (PXN) binding . This post-translational modification affects how ITGA4-containing integrins (particularly α4β1/VLA-4 and α4β7) interact with extracellular matrix components like fibronectin and cell adhesion molecules such as VCAM1 and MADCAM1. These interactions are crucial for leukocyte trafficking, homotypic cell aggregation, and potentially cytolytic T-cell interactions with target cells . Understanding this phosphorylation event is particularly important when studying immune cell function, cancer cell invasion, and inflammatory disease processes.
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How should researchers prepare samples for optimal detection of phosphorylated ITGA4?
For optimal detection of phosphorylated ITGA4, follow these methodological guidelines:
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For IHC applications: Perform antigen retrieval using sodium citrate (10 mmol/l, pH 6.0) in an autoclave (120°C for 2 min) . For paraffin sections, high-pressure and temperature Tris-EDTA (pH 8.0) has also proven effective for antigen retrieval .
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Sample preservation: Store tissues properly before immunostaining; flash-frozen samples or tissues fixed in 10% neutral-buffered formalin and embedded in paraffin are recommended.
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Blocking step: Use appropriate blocking solutions (typically containing 0.5% BSA) to minimize non-specific binding .
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Phosphatase inhibitors: Include phosphatase inhibitors during sample preparation to prevent dephosphorylation of the target epitope.
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Storage of antibody: Store the antibody at -20°C for up to one year from receipt date, and avoid repeat freeze-thaw cycles .
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What controls should be included when using Phospho-ITGA4 (S1027) antibodies?
When using Phospho-ITGA4 (S1027) antibodies, include the following controls to ensure experimental validity:
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Positive control: Use cell lines or tissues known to express phosphorylated ITGA4 (Jurkat T cells are often used for this purpose) .
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Negative control: Include antibody pre-absorbed by the immunogen peptide. This has been demonstrated in immunohistochemical analysis where the negative control showed no staining when the antibody was pre-absorbed with the immunogen peptide .
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Phosphatase treatment control: Treat a portion of your sample with lambda phosphatase before antibody incubation to confirm phospho-specificity.
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siRNA or gene knockout control: When available, use ITGA4 knockdown or knockout samples as additional specificity controls .
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Isotype control: Include an irrelevant antibody of the same isotype (typically rabbit IgG) to account for non-specific binding .
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