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- Order number: 7TM0071N
- Content: 100 µl
- Host: Rabbit
The non-phospho-CXCR4 receptor antibody is directed against the distal end of the carboxyl-terminal tail of mouse, rat and human CXCR4. It can be used to detect CXCR4 receptors in Western blots in a phosphorylation-sensitive manner. After agonist activation CXCR4 is phosphorylated at S346/S347. Because the CXCR4 antibody detects the same epitope, it no longer binds to the receptor. It detects selectively CXCR4 receptors that have been hot activated and not phosphorylated. Total CXCR4 can be detected in Western blots and immunohistochemistry only after phosphatase treatment. It can also be used to isolate and enrich CXCR4 receptors from brain lysates. It also detects CXCR4 in cultured cells and tissue sections by immunohistochemistry. The non-phospho-CXCR4 antibody has been validated using knockout mice (KO-Validated).
| Alternative Names | CXCR4, CXC Chemokin Receptor 4 |
| IUPHAR Target ID | 71 |
| UniProt ID | P61073 (human) P70658 (mouse) O08565 (rat) |
| Western Blot (WB) | 1:1000 |
| Immunocytochemistry (ICC) | 1:200 |
| Immunohistochemisty (IHC) | 1:100 |
| Species Reactivity | Human, Mouse, Rat |
| Host / Isotype | Rabbit / IgG |
| Class | Polyclonal |
| Immunogen | A synthetic peptide with the sequence KGKRGGHSSVSTESESSSFHSS corresponding to residues 338-359 in human, mouse and rat CXCR4 |
| Form | Liquid |
| Purification | Antigen affinity chromatography |
| Storage buffer | Dulbecco's PBS, pH 7.4, with 150 mM NaCl, 0.02% sodium azide |
| Storage conditions | short-term 4°C, long-term -20°C |
Figure 1. Validation of the CXC Chemokine Receptor 4 in transfected HEK293 cells. Native HEK293 cells (MOCK) or HEK293 cells stably expressing the CXC Chemokine Receptor 4 (CXCR4) were lysed and immunoblotted with the phosphorylation-sensitive anti-CXCR4 antibody (7TM0071N-WB) at a dilution of 1:1000.
Figure 2. Western blot analysis of CXC Chemokine Receptor 4 in mouse brain in vivo. Brains from CXCR4-deficient mice (CXCR4-KO) and their wild-type littermates (CXCR4-WT) were dissected, homogenated and immunoblotted with anti-CXCR4 (non-phospho-CXC Chemokine receptor 4) antibody (7TM0071N-WB) at a dilution of 1:1000. Note, absence of CXCR4 receptors in CXCR4-KO but not in CXCR4-WT mice.
Figure 3. Immunohistochemical identification of CXC Chemokine Receptor 4 receptor in mouse cerebral cortex. Sections of mouse E17 forebrains were dewaxed, microwaved in citric acid, and incubated with anti-CXCR4 (non-phospho-CXC Chemokine receptor 4 receptor) antibody (7TM0071N-WB) at a dilution of 1:100. Sections were then sequentially treated with biotinylated anti-rabbit IgG and avidin-biotin solution. Sections were then developed in 3,3-diaminobenzidine (DAB)-glucose oxidase and lightly counterstained with hematoxylin. Note, CXCR4 receptors were detected at the plasma membrane of tangentially migrating neurons in the cerebral cortex.
Figure 4. Analysis of dose-dependent Serine346/Serine347 CXC Chemokine Receptor 4 phosphorylation. Upper panel, HEK293 cells stably expressing the HA-tagged CXC Chemokine Receptor 4 were either not exposed or exposed to increasing concentrations of CXCL12 ranging from 1 nM to 100 nM for 30 minutes. Cells were lysed and immunoblotted with the anti-pS346/pS347-CXCR4 antibody (7TM0071B) at a dilution of 1:1000. Middle panel, blot was stripped and reprobed with the phosphorylation-sensitive anti-CXCR4 antibody (7TM0071N-WB). Lower panel, blot was stripped and reprobed with the phosphorylation-independent anti-HA antibody (7TM000HA) at a dilution of 1:1000 to confirm equal loading of the gel.
Figure 5. Immunohistochemical identification of CXC Chemokine Receptor 4 receptor in human pheochromacytoma. Sections were dewaxed, microwaved in citric acid, and incubated with anti-CXCR4 (non-phospho-CXC Chemokine Receptor 4) antibody (7TM0071N-WB) at a dilution of 1:100. Sections were then sequentially treated with biotinylated anti-rabbit IgG and avidin-biotin solution. Sections were then developed in 3,3-diaminobenzidine (DAB)-glucose oxidase and lightly counterstained with hematoxylin. Note, CXCR4 receptors were uniformly detected at the plasma membrane of nearly all tumor cells.
Figure 6. Immunohistochemical identification of CXC Chemokine Receptor 4 in human endometrial carcinoma. Sections were dewaxed, microwaved in citric acid, and incubated with anti-CXCR4 (non-phospho-CXC Chemokine Receptor 4) antibody (7TM0071N-WB) at a dilution of 1:100. Sections were then sequentially treated with biotinylated anti-rabbit IgG and avidin-biotin solution. Sections were then developed in 3,3-diaminobenzidine (DAB)-glucose oxidase and lightly counterstained with hematoxylin. Note, CXCR4 receptors were uniformly detected at the plasma membrane of nearly all tumor cells.
Figure 7. Immunocytochemical identification of CXC Chemokine Receptor 4 in HEK293 cells. HEK293 cells stably expressing the CXC Chemokine Receptor 4 (CXCR4) were either not exposed or exposed to 100 nM CXCl-12 for 30 min and immunocytochemically stained with anti-CXCR4 (non-phospho-CXC Chemokine Receptor 4) antibody (7TM0071N-WB) at a dilution of 1:200. Note, CXCR4 receptors were confined to the plasma membrane in untreated cells (0 min).CXCR4 receptors were seen in perinuclear clusters of vesicles after 30 min CXCL-12 exposure.
