Figure 1: CXCR4 target protein structure.

CXCR4 Target Introduction

Protein Function

• Receptor for the C-X-C chemokine CXCL12/SDF-1, involved in transducing signals by increasing intracellular calcium levels and enhancing MAPK1/MAPK3 activation.
• Involved in AKT signaling cascade.
• Plays a regulatory role in cell migration, such as wound healing.
• Acts as a receptor for extracellular ubiquitin, leading to increased intracellular calcium and decreased cellular cAMP levels.
• Binds to bacterial lipopolysaccharide (LPS) and mediates LPS-induced inflammatory response, including monocyte secretion of TNF.
• Involved in hematopoiesis and ventricular septum formation, also plays an essential role in vascularization of the gastrointestinal tract, possibly through regulating vascular branching and/or endothelial cell remodeling processes; involved in cerebellar development; in the central nervous system, mediates survival of hippocampal neurons.
• Serves as a co-receptor for human immunodeficiency virus-1 (HIV-1) X4 strains (with CD4 as the central receptor) and as the primary receptor for certain HIV-2 strains, facilitating Env-mediated virus fusion.

Protein Expression

• Expressed in various tissues, including peripheral blood leukocytes, spleen, thymus, spinal cord, heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, cerebellum, cerebral cortex, and white matter (oligodendrocytes and astrocytes), cerebral microvessels, coronary arteries, and umbilical cord endothelial cells.
• The most predominantly expressed isoform in all tested tissues is isoform 1.

Protein Localization

• 7-transmembrane protein, localized to the cell membrane, cell junctions, early endosomes, late endosomes, and lysosomes.
• In unstimulated cells, it is distributed diffusely on the cell membrane. Upon stimulation by agonists, it co-localizes with ITCH at the cell membrane, leading to its ubiquitination. In the presence of antigens, it is distributed at the T cell-APC contact area, forming an immunological synapse and localizes to the peripheral and distal supramolecular activation clusters (SMACs).

Figure 2: CXCR4 ICC experimental result image, Anti-CXCR4 antibody [UMB2] (ab124824). Green: CXCR4; Red: Tubulin; Blue: DAPI.

Isoforms & Post-translational modifications

Human (P61073): Isoform 1-2: 39-40 kDa (predicted)
Mouse (P70658): Isoform 1-2: 40 kDa (predicted)
Rat (O08565): 39 kDa (predicted)
Existence of post-translational modifications such as phosphorylation, disulfide bonding, glycosylation, ubiquitination, etc.

WB experiment tips

Precautions

• CXCR4 is a multi-pass transmembrane protein, and high-temperature boiling can cause protein aggregation. If the protein becomes larger, has a tail, or has no signal after boiling, we recommend not boiling the sample after lysis.
• If the signal is weak in the whole cell lysate, try extracting the cell membrane fraction to enrich the target protein.
• To avoid the impact of phosphorylation on CXCR4 detection, we recommend treating the membrane with λ protein phosphatase before incubating with the primary antibody.

Positive control

• Jurkat whole cell lysate

Example of results

Figure 3: WB-Anti-CXCR4 antibody [UMB2] (ab124824).

Lane 1: 15 µg HeLa whole cell lysate.
Lanes 2-3: 15 µg Jurkat whole cell lysate.
Predicted band size: 39 kDa.
Actual band size: 43 kDa.
Note: We recommend not boiling the samples after lysis.

Figure 4: WB-Anti-CXCR4 antibody [UMB2] (ab124824).

Lane 1: 20 µg CHO whole cell lysate (negative control)
Lane 2: 20 µg Jurkat whole cell lysate
Lane 3: 20 µg Jurkat cell membrane extract
Lane 4: 20 µg Jurkat cell nuclear extract (negative control)

Predicted band size:  39 kDa
Actual band size: 41 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex proteinase inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.

Transferring:

1. We recommend using Ponceau S staining after transferring to determine the success of the transfer.

References

1. S K Gupta, K Pillarisetti. Cutting edge: CXCR4-Lo: molecular cloning and functional expression of a novel human CXCR4 splice variant. Comparative Study J Immunol. 1999 Sep 1;163(5):2368-72.
2. Y Cao, Z R Hunter, X Liu, L Xu, G Yang, J Chen, C J Patterson, N Tsakmaklis, S Kanan, S Rodig, J J Castillo, S P Treon. The WHIM-like CXCR4(S338X) somatic mutation activates AKT and ERK, and promotes resistance to ibrutinib and other agents used in the treatment of Waldenstrom's Macroglobulinemia. Leukemia. 2015 Jan;29(1):169-76. doi: 10.1038/leu.2014.187. Epub 2014 Jun 10.
3. Travis Lear, Sarah R Dunn, Alison C McKelvey, Aazrin Mir, John Evankovich, Bill B Chen, Yuan Liu. RING finger protein 113A regulates C-X-C chemokine receptor type 4 stability and signaling. Am J Physiol Cell Physiol. 2017 Nov 1;313(5):C584-C592. doi: 10.1152/ajpcell.00193.2017. Epub 2017 Oct 4.
4. Vikas Saini, Adriano Marchese, Matthias Majetschak. CXC chemokine receptor 4 is a cell surface receptor for extracellular ubiquitin. J Biol Chem. 2010 May 14;285(20):15566-15576. doi: 10.1074/jbc.M110.103408. Epub 2010 Mar 12.
5. K Triantafilou, M Triantafilou, R L Dedrick. A CD14-independent LPS receptor cluster. Nat Immunol. 2001 Apr;2(4):338-45. doi: 10.1038/86342.
6. C K Lapham, J Ouyang, B Chandrasekhar, N Y Nguyen, D S Dimitrov, H Golding. Evidence for cell-surface association between fusin and the CD4-gp120 complex in human cell lines. Science. 1996 Oct 25;274(5287):602-5. doi: 10.1126/science.274.5287.602.