ATP1B3 Antibody

What is ATP1B3 and what is its biological function?

ATP1B3, also known as CD298, is the β3 subunit of Na+/K+-ATPase which functions to maintain sodium and potassium gradients across membranes involved in cellular activities . This protein is critical for:

• Maintaining electrochemical gradients across cell membranes

• Supporting cellular homeostasis

• Enabling proper functioning of ion transport mechanisms

• Contributing to cell adhesion and signaling processes

ATP1B3 is a glycosylated protein, with both fully and intermediately glycosylated forms present in mammalian cells . The protein consists of a cytoplasmic domain (residues 1-35), a helical domain (residues 36-56), and an extracellular domain (residues 57-279) .

What is the molecular weight of ATP1B3 and why does it vary in experimental observations?

ATP1B3 exhibits an interesting discrepancy between its calculated and observed molecular weights:

This variation is attributed to different levels of glycosylation of the protein . Glycosylation is a post-translational modification that adds sugar moieties to proteins, increasing their molecular weight. The observed 38-43 kDa range reflects various glycosylated forms of ATP1B3 detected in experimental settings.

In which cell types and tissues is ATP1B3 typically expressed?

ATP1B3 shows expression across multiple cell types and tissues as evidenced by antibody validation data:

Expression has been particularly noted in rat lung and testis according to some studies .

What are the validated applications for ATP1B3 antibodies?

ATP1B3 antibodies have been validated for multiple experimental applications:

For optimal results in immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may also be used as an alternative .

What are the optimal storage conditions for ATP1B3 antibodies?

Proper storage is critical for maintaining antibody integrity and performance:

• Store at -20°C for long-term preservation

• Antibodies are typically stable for one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage

• Antibodies are typically shipped at 4°C

• Avoid repeated freeze/thaw cycles to maintain antibody activity

Most commercial ATP1B3 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during storage .

How does ATP1B3 contribute to cancer biology?

Recent research has identified significant roles for ATP1B3 in various cancer types:

Hepatocellular Carcinoma (HCC)

ATP1B3 expression is significantly associated with HCC progression through:

Glioma

ATP1B3 has been identified as a potential therapeutic target in glioma research:

• Expression correlation: ATP1B3 expression positively correlates with glioma grade

• Functional impact: Knockdown of ATP1B3 in glioma cell lines (U87MG and U251MG) significantly reduces cell proliferation, migration, and invasion capabilities

• Signaling pathways: ATP1B3 may promote glioma progression by regulating the MAPK and NF-κB signaling pathways

• Protein interactions: ATP1B3 indirectly regulates the downstream protein PPP1CA

What role does ATP1B3 play in immune regulation?

ATP1B3 exhibits significant correlations with immune markers and cytokines:

• Positive correlations with markers of:

  • CD8+ T cells

  • General T cells

  • M1 Macrophages

  • B cells

  • Tumor-associated macrophages (TAM)

  • Dendritic cells (DCs)

  • T helper cell 1 (Th1)

  • Follicular helper T cells (Tfh)

  • T cell exhaustion

• Cytokine associations:

  • Positive correlation with IL10, IL22, and IL34

  • Negative correlation with IL27

These interactions suggest potential roles in the immune microenvironment:

• IL10 can inhibit NK cell cytotoxicity through STAT3 signaling, potentially promoting cancer metastasis

• IL22 is often highly expressed in HCC and relates to tumor growth and malignancy

• IL34 promotes proliferation and migration of HCC through CSF1-R and CD138

• IL27, which negatively correlates with ATP1B3, can exert anti-tumor activity by activating NK cells

How does ATP1B3 influence viral infections like HIV-1?

ATP1B3 has been identified as a novel BST-2-binding protein with implications for HIV-1 infection:

• ATP1B3 depletion in BST-2-positive cells (HeLa, THP-1) reduces HIV-1 p24 production

• This effect is not observed in BST-2-negative cells (293T), suggesting a BST-2-dependent mechanism

• The interaction between ATP1B3 and BST-2 has been confirmed through co-immunoprecipitation experiments

• ATP1B3 knockdown in BST-2-expressing cells impairs HIV-1 production

These findings suggest ATP1B3 may regulate BST-2's restriction of HIV-1, representing a potential therapeutic target for HIV research.

What are the key considerations for optimizing Western blot detection of ATP1B3?

For effective Western blot detection of ATP1B3:

• Sample preparation: ATP1B3 has been successfully detected in lysates from various cell types including Jurkat, THP-1, HeLa, and HepG2 cells

• Antibody dilution: Use dilutions between 1:500-1:4000 depending on the specific antibody and sample type

• Expected band size: Look for bands at 38-43 kDa rather than the calculated 32 kDa due to glycosylation

• Multiple bands: Be aware that different glycosylation states may result in multiple bands

• Controls: Use positive control lysates like Jurkat or THP-1 cells, which consistently show strong ATP1B3 expression

How can researchers optimize immunohistochemical detection of ATP1B3?

For optimal immunohistochemical detection:

• Antigen retrieval: Use TE buffer at pH 9.0 as the primary option, with citrate buffer at pH 6.0 as an alternative

• Antibody dilution: Use between 1:50-1:2000 depending on the specific antibody and tissue type

• Positive control tissues: Human prostate cancer, liver cancer, colon, and lung cancer tissues have shown reliable ATP1B3 expression

• Blocking peptides: Consider using ATP1B3-specific blocking peptides as negative controls to confirm staining specificity

• Detection systems: Both chromogenic and fluorescent secondary detection systems have been validated

What approaches can be used to study ATP1B3 function in experimental models?

Several validated approaches have been used to investigate ATP1B3 function:

• RNA interference: siRNA-mediated knockdown (e.g., si-ATP1B3-336) has been successfully used to reduce ATP1B3 expression in various cell lines

• Functional assays following knockdown:

  • Proliferation assays to assess growth effects

  • Migration and invasion assays to evaluate cell motility

  • Viral production assays to examine effects on HIV-1 replication

• Protein interaction studies:

  • Co-immunoprecipitation to identify binding partners like BST-2

  • Construction of deletion mutants to map binding domains

• Signaling pathway analysis:

  • Examination of effects on MAPK pathway components (p-Raf1, p-MEK 1/2, p-ERK 1/2)

  • Analysis of NF-κB pathway components (p-IκBα, p-P65)

  • Downstream effects on regulatory proteins like Cyclin D1 and VEGFA

How can researchers address discrepancies in detected molecular weight?

When encountering unexpected band sizes:

• Remember that while the calculated molecular weight of ATP1B3 is 32 kDa, observed bands typically range from 38-43 kDa due to glycosylation

• Multiple bands may represent different glycosylation states of the protein

• Consider using deglycosylation enzymes to confirm that higher molecular weight bands are indeed glycosylated forms of ATP1B3

• Compare results across multiple cell lines with known ATP1B3 expression (e.g., Jurkat, THP-1)

• Verify antibody specificity using knockdown controls or blocking peptides when available

What controls should be included in ATP1B3 expression and functional studies?

For robust experimental design:

• Positive expression controls: Include cells with known ATP1B3 expression (Jurkat, THP-1, HeLa)

• Negative expression controls: Consider using cells with low or no ATP1B3 expression when applicable

• Knockdown validation: Confirm ATP1B3 reduction at both mRNA (RT-qPCR) and protein (Western blot) levels

• Multiple siRNA sequences: Use different siRNA sequences targeting ATP1B3 to rule out off-target effects

• Rescue experiments: Reintroduce wild-type ATP1B3 to determine if observed phenotypes can be reversed

• Isotype controls: Include appropriate isotype controls for immunostaining experiments