TNPO3 Antibody

What is TNPO3 and what cellular functions does it perform?

TNPO3 (Transportin-3) is a karyopherin that functions primarily as a nuclear import receptor. In cellular contexts, TNPO3 facilitates the transport of proteins between the cytoplasm and nucleus. Research demonstrates that TNPO3 plays critical roles in multiple biological pathways:

• B cell development: TNPO3 interacts with EBF1 (Early B-cell Factor 1) through EBF1's immunoglobulin-like fold domain, particularly at glutamic acid 271. This interaction is essential for proper B cell programming under conditions where T-lineage promoting Notch signaling is present. The association occurs in the nucleus and enables efficient B cell development in nonpermissive conditions .

• T cell differentiation: Previous studies indicate that TNPO3 participates in early T cell differentiation processes, with its deletion resulting in partial blocks of T cell development and reduced TCR signaling in CD8+ T cells .

• Viral replication: TNPO3 is required for the replication of several retroviruses, including HIV-1, HIV-2, SIVmac, EIAV, and BIV, though its exact mechanism varies among different viral systems .

Detection of TNPO3 via antibodies typically reveals subcellular distribution patterns with predominant localization in cytoplasmic fractions, though nuclear presence is also detected during specific interactions .

What experimental techniques are optimal for detecting TNPO3 using antibodies?

For effective detection of TNPO3 protein using antibodies, researchers should consider the following experimental approaches:

Western Blotting:

• Use whole-cell extracts or fractionated cell preparations (nuclear vs. cytoplasmic)

• TNPO3 protein can be detected in both nuclear and cytoplasmic fractions, though studies show predominant detection in cytoplasmic fractions

• Recommended controls: Include both positive control (known TNPO3-expressing cells) and negative control (TNPO3 knockdown cells)

Co-immunoprecipitation (Co-IP):

• Effective for studying TNPO3 interactions with partner proteins

• Successfully used to confirm TNPO3 interaction with EBF1 in both whole-cell and nuclear extracts of pro-B cells

• For viral studies, can be used to analyze TNPO3 interactions with viral components like HIV-1 capsid proteins

Immunofluorescence:

• Useful for visualizing subcellular localization

• Consider dual staining with nuclear markers to assess import/export functions

• Controls should include TNPO3 knockdown cells to confirm antibody specificity

Quantitative techniques:

• SILAC-based mass spectrometry has been successfully employed alongside immunoprecipitation to identify TNPO3-interacting proteins with high sensitivity

How can I validate the specificity of a TNPO3 antibody?

Validating TNPO3 antibody specificity is critical for ensuring reliable experimental results. Recommended validation approaches include:

Genetic validation:

• Generate TNPO3 knockdown cells using siRNA or shRNA approaches

• Compare Western blot signals between control and knockdown samples

• A specific antibody will show significantly reduced signal in knockdown cells

Rescue experiments:

• After TNPO3 knockdown, express a non-targetable TNPO3 cDNA (ntTNPO3) containing silent mutations that render it resistant to the knockdown

• Confirm restored antibody signal with the rescue construct

Multiple antibody concordance:

• Use antibodies targeting different epitopes of TNPO3

• Consistent results across different antibodies support specificity

Recombinant protein control:

• Use purified recombinant TNPO3 protein as a positive control

• This approach has been used in binding studies with HIV-1 CA-NC complexes

Example validation strategy from literature:
Researchers have successfully used sequential transduction with knockdown and rescue vectors, followed by selection with different antibiotics (puromycin and blasticidin) to generate cellular systems for validating TNPO3 antibody specificity .

What controls should be included when using TNPO3 antibodies in functional studies?

When conducting functional studies with TNPO3 antibodies, include these essential controls:

Cellular controls:

• Positive control: Cell lines known to express TNPO3 (e.g., HeLa, 38B9 pro-B cells)

• Negative control: TNPO3 knockdown cells generated using validated siRNA or shRNA constructs

• Rescue control: Cells expressing non-targetable TNPO3 to confirm function restoration

Antibody controls:

• Isotype control: Use species-matched, non-specific antibody

• Secondary antibody only: Ensure no non-specific binding

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

Functional assay controls:

• For nuclear import studies: Include importin-β inhibitors as comparative controls

• For viral infection studies: Include control viruses (e.g., MMLV or FIV) that show TNPO3-independent replication

• For protein interaction studies: Include non-interacting protein controls and mutation controls (e.g., EBF1 E271A which disrupts TNPO3 interaction)

How can TNPO3 antibodies be used to study protein-protein interactions?

TNPO3 antibodies are valuable tools for investigating protein-protein interactions through multiple complementary approaches:

Co-immunoprecipitation (Co-IP):

• Use TNPO3 antibodies to pull down TNPO3 and associated proteins

• This approach successfully identified the interaction between TNPO3 and EBF1 in pro-B cells

• Protocol recommendation: Perform Co-IP in both whole-cell extracts and nuclear extracts to determine compartment-specific interactions

Reciprocal Co-IP:

• Use antibodies against suspected binding partners to pull down complexes

• Probe with TNPO3 antibody to confirm interaction

• Example: Researchers confirmed EBF1-TNPO3 interaction using both approaches

Combined with mutational analysis:

• Generate mutant constructs of the interacting partner

• TNPO3 interaction with EBF1 was mapped by creating domain deletion mutants and point mutations

• Glutamic acid 271 in the EBF1 IPT domain was identified as critical for TNPO3 interaction

SILAC-MS approach:

• Label cells expressing wild-type or mutant proteins with heavy or light amino acids

• Immunoprecipitate with specific antibodies

• Analyze differential protein associations by mass spectrometry

• This approach confirmed that Tnpo3 was the only protein showing differential interaction between EBF1 wild-type and E271A mutant

Example experimental scheme:

• Transfect cells with wild-type and mutant constructs

• Perform Co-IP using TNPO3 antibody

• Analyze precipitated proteins by Western blot or mass spectrometry

• Validate interactions using reciprocal Co-IP

• Confirm specificity using TNPO3 knockdown cells

What methodological approaches can reveal TNPO3's role in HIV-1 infection?

Investigating TNPO3's function in HIV-1 infection requires targeted experimental strategies:

TNPO3 depletion studies:

• Generate stable TNPO3 knockdown cell lines using shRNA

• Measure HIV-1 infectivity using reporter viruses (e.g., GFP-expressing HIV-1)

• Studies show that TNPO3 depletion reduces HIV-1 infection by approximately 12-fold

Comparative viral susceptibility analysis:

• Test multiple retroviruses in TNPO3 knockdown cells

• Research demonstrates differential effects: strong inhibition of SIVmac (17-fold), HIV-2 (15-fold), and HIV-1 (12-fold), moderate effects on BIV (4-fold) and EIAV (3-fold), and no effect on MMLV or FIV

Mechanistic analysis with qPCR:

• Use qPCR to measure viral DNA products at different stages:

  • Early reverse transcription products

  • Late reverse transcription products

  • 2-LTR circles (marker of nuclear entry)

  • Integrated proviral DNA

• Studies show TNPO3 depletion blocks HIV-1 replication after nuclear import but prior to integration

Capsid binding assays:

• Use in vitro-assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes

• Incubate with purified TNPO3

• Sediment complexes through sucrose cushion

• Analyze binding by Western blot

• Data indicates TNPO3 binds directly to HIV-1 CA-NC complexes in a concentration-dependent manner

Capsid mutant analysis:

• Generate HIV-1 vectors with different CA mutations

• Test their dependency on TNPO3 for infection

• Specific mutations (e.g., N74D) render HIV-1 less sensitive to TNPO3 depletion

• In vitro binding assays confirm reduced affinity of TNPO3 for N74D mutant capsids

How do I optimize experimental protocols for studying TNPO3 interactions with viral components?

To effectively study TNPO3 interactions with viral components, consider these methodological optimizations:

In vitro binding assays:

• Purify recombinant TNPO3 protein

• Assemble viral CA-NC complexes in vitro

• Incubate in binding buffer with increasing TNPO3 concentrations

• Separate bound complexes using sucrose cushion ultracentrifugation

• Analyze by Western blot using TNPO3 antibodies

• Critical controls: Include TNPO3 in cellular extracts without CA-NC complexes as negative control

Mutational analysis protocol:

• Generate viral vectors with capsid mutations (panel of 27 different CA mutants used in one study)

• Test infectivity in TNPO3 knockdown cells

• Compare to wild-type virus

• Identify mutations that alter TNPO3 dependency

• Follow-up with binding assays for key mutants

Nuclear import analysis:

• Fractionate cells into cytoplasmic and nuclear components

• Use Western blotting with TNPO3 antibodies to assess distribution

• Analyze viral components in each fraction

• Also consider immunofluorescence microscopy for visualization

Time-course experiments:

• Synchronize infection

• Harvest cells at multiple time points

• Analyze viral DNA products (RT products, 2-LTR circles, integrated DNA)

• Include appropriate controls:

  • RT inhibitors (e.g., nevirapine)

  • Integrase inhibitors (e.g., raltegravir)

  • TNPO3 rescue cells

What is the significance of TNPO3 in B cell development and how can antibodies help investigate this function?

TNPO3 plays a critical role in B cell development through its interaction with the transcription factor EBF1. This function can be investigated using TNPO3 antibodies through several approaches:

Mechanistic investigation:

• TNPO3 interacts with EBF1 via the immunoglobulin-like fold domain, specifically requiring glutamic acid 271

• This interaction is essential for B cell programming in nonpermissive conditions with T-lineage promoting Notch signaling

• TNPO3 does not affect EBF1 nuclear localization but enhances its function

Experimental approaches:

• B cell-specific TNPO3 knockout studies:

  • Generate B cell-specific TNPO3 knockout mice

  • Analyze B cell differentiation in bone marrow

  • Assess expression of B cell-specific EBF1 target genes

  • Measure expression of T cell lineage-associated genes

  • Research shows TNPO3 deletion blocks early B cell differentiation and increases T cell gene expression

• Gene expression analysis:

  • Use TNPO3 antibodies to confirm knockdown or knockout

  • Perform RNA-seq or qPCR to assess effects on B cell-specific genes

  • Analyze EBF1 target gene expression in presence/absence of TNPO3

• Chromatin immunoprecipitation (ChIP):

  • Use TNPO3 antibodies for ChIP to assess potential chromatin association

  • Compare EBF1 binding to target genes with and without TNPO3

• Co-localization studies:

  • Use immunofluorescence with TNPO3 antibodies and EBF1 antibodies

  • Assess nuclear co-localization

  • Compare wild-type and E271A mutant EBF1

Impact of results:
Understanding TNPO3's role in B cell development provides insights into lineage commitment mechanisms and may have implications for B cell malignancies and immune disorders.

How can I distinguish between TNPO3's role in nuclear import versus other functions when using TNPO3 antibodies?

TNPO3 is primarily known as a nuclear import receptor, but research indicates additional functions. To distinguish between these roles using TNPO3 antibodies:

Experimental approaches:

• Subcellular fractionation analysis:

  • Separate cells into cytoplasmic, nucleoplasmic, and chromatin-bound fractions

  • Use TNPO3 antibodies to detect distribution across fractions

  • Compare with known nuclear import substrates

  • Research shows TNPO3 is predominantly detected in cytoplasmic fractions

• Domain-specific mutant analysis:

  • Generate TNPO3 constructs with mutations in:

    • Ran-binding domain (affects nuclear import function)

    • Cargo-binding domain (CBD)

  • Test functionality of each mutant

  • Analyze protein interactions using TNPO3 antibodies

  • Studies of RSV Gag show that nuclear entry does not require the CBD of TNPO3

• Time-course immunofluorescence:

  • Follow TNPO3 localization during cellular processes

  • Co-stain with interaction partners

  • Analyze dynamics during viral infection

• Interaction mapping:

  • Use TNPO3 antibodies for immunoprecipitation followed by mass spectrometry

  • Compare binding partners in different cellular compartments

  • Identify proteins that interact with TNPO3 in unusual locations

• Functional complementation:

  • In TNPO3 knockdown cells, express:

    • Wild-type TNPO3

    • Nuclear import-deficient TNPO3

    • Other domain-specific mutants

  • Test rescue of different functions

  • Use TNPO3 antibodies to confirm expression levels

Distinguishing features in retroviral systems:

• TNPO3 depletion blocks HIV-1 replication after nuclear import but prior to integration

• This suggests functions beyond simple nuclear import

• In contrast, for RSV Gag, TNPO3 directly facilitates nuclear entry