NUP133 Antibody

What is NUP133 and why is it significant in cellular research?

NUP133 (Nucleoporin 133) is a crucial component of the nuclear pore complex (NPC) with a molecular mass of approximately 129 kDa. In humans, the canonical protein consists of 1156 amino acid residues and is primarily localized in the nucleus. NUP133 plays a significant role in the transport of macromolecules between the nucleus and cytoplasm, which is vital for maintaining cellular function and gene expression regulation .

NUP133 research is particularly significant because:

• It is widely expressed in both fetal and adult tissues

• It belongs to the Nucleoporin Nup133 protein family involved in poly(A)+ RNA transport

• It has been associated with nephrotic syndrome

• It is strategically located on both cytoplasmic and nuclear sides of the nuclear pore

• During mitosis, it localizes to kinetochores, highlighting its importance in cell division

The protein is part of the Nup160 nuclear pore subcomplex, which includes Nup160, Nup96, and Nup107, and is essential for RNA export, ensuring that mRNA and other RNA species are efficiently transported out of the nucleus to the cytoplasm for translation .

How is NUP133 structurally organized and what are its functional domains?

NUP133 contains distinct structural domains with specific functions:

N-terminal domain (NTD):

• Contains a β-propeller structure

• Features an amphipathic lipid packing sensor (ALPS) motif in the DA 34 loop (residues 252-270 in yeast), which can bind to and modify curved biological membranes

• Is responsible for membrane association and plays a role in nuclear pore complex assembly

• Interacts with Cenp-F through a conserved helix within the β-propeller

C-terminal domain (CTD):

• Forms a complex with Nup84

• Is part of the Y complex, an essential scaffolding component of the NPC

• Shows high flexibility, which is important for NPC structure and function

The entire protein contains distinctive features including O-linked N-acetylglucosamine moieties and a pentapeptide repeat (XFXFG), which are important for nucleocytoplasmic transport regulation .

What criteria should researchers consider when selecting the appropriate NUP133 antibody for their experimental needs?

When selecting a NUP133 antibody, researchers should consider several key factors:

Species reactivity and cross-reactivity:

• Determine which species your samples come from and select antibodies validated for those species

• Common validated species include human, mouse, rat, and other model organisms

• Consider evolutionary conservation of NUP133 when working with non-standard model organisms

Antibody format and conjugation:

• Unconjugated antibodies for flexible detection methods

• Pre-conjugated options (HRP, PE, FITC, Alexa Fluor conjugates) for direct detection

• Agarose-conjugated for immunoprecipitation applications

Application compatibility:

• Western Blot (WB): Most NUP133 antibodies are validated for WB

• Immunoprecipitation (IP): Select antibodies specifically validated for IP

• Immunofluorescence (IF): Crucial for localization studies

• ELISA: Important for quantitative studies

• Immunohistochemistry (IHC): Consider tissue-specific validation

Target epitope location:

• N-terminal specific antibodies: Useful for studying NUP133 membrane interactions

• C-terminal specific antibodies: Better for studying interactions with Nup84 complex

• Middle region antibodies: May detect a wider range of NUP133 variants

Validation data:

• Check for published citation records with the antibody

• Review available images showing expected cellular localization

• Examine bandwidths in Western blot validations (expected at ~129-133 kDa)

• Consider antibodies with knockout/knockdown validation

How should researchers validate a new NUP133 antibody for their specific experimental system?

A robust validation strategy for NUP133 antibodies should include:

1. Western blot validation:

• Expected molecular weight: 129-133 kDa

• Positive control tissues/cell lines: A375, LO2, HeLa, NCI-H460

• Test antibody specificity with recommended dilutions (typically 1:1000-1:2000)

• Compare against existing validated antibodies if available

2. Subcellular localization confirmation:

• Immunofluorescence should show nuclear envelope staining pattern

• Co-staining with other nuclear pore markers like Nup107 or Nup98

• During mitosis, should show kinetochore localization

3. Functional validation:

• Immunoprecipitation followed by mass spectrometry to confirm interaction partners (should pull down other Y complex components)

• Confirmation of antibody-target binding through knockout/knockdown controls

• Super-resolution microscopy visualization of NPC localization

4. Cross-validation with genetic approaches:

• Use in cells with Nup133 depletion (siRNA, CRISPR, etc.)

• Test in Nup133−/− mESCs as a negative control

• Rescue experiments with NUP133 expression to restore staining pattern

What are the optimal experimental conditions for studying NUP133 localization during different cell cycle phases?

Studying NUP133 localization across the cell cycle requires specific experimental considerations:

Cell cycle synchronization methods:

• G1/S arrest: Double thymidine block

• G2/M arrest: Nocodazole treatment

• Prophase: RO-3306 (CDK1 inhibitor) release

• Metaphase: MG132 (proteasome inhibitor)

• Anaphase/Telophase: Release from metaphase arrest

Immunofluorescence protocol optimization:

• Fixation: 4% paraformaldehyde preserves nuclear structure

• Permeabilization: 0.1-0.5% Triton X-100 allows antibody access to nuclear pores

• Blocking: 3-5% BSA to reduce background

• Primary antibody dilution: Typically 1:100-1:500 for most NUP133 antibodies

• Co-staining markers:

  • DNA (DAPI/Hoechst)

  • Microtubules (α-tubulin)

  • Kinetochores (CENP-B, Hec1)

  • Other NPC components (Nup107, Nup98, Tpr)

Advanced imaging approaches:

• Super-resolution microscopy (SIM, STORM, STED) to resolve individual pore structures

• Live-CLEM (Correlative Light and Electron Microscopy) for dynamic studies

• Fluorescence recovery after photobleaching (FRAP) to study dynamics

• Recommended microscope settings: 60-100x objectives with high NA, deconvolution for optimal resolution

Research findings show that NUP133 exhibits differential localization during cell cycle progression:

• Interphase: Nuclear envelope staining with distinct punctate pattern at nuclear pores

• Prophase: Begins to associate with kinetochores while remaining at fragmenting nuclear envelope

• Metaphase: Primarily at kinetochores with diffuse cytoplasmic pool

• Telophase: Reassociates with reforming nuclear envelope through membrane fenestrations

What experimental approaches can determine if NUP133 is correctly assembled into the nuclear pore complex?

Several complementary approaches can assess NUP133 assembly status:

Biochemical approaches:

• Co-immunoprecipitation with other Y complex components (Nup107, Nup160, Nup96)

• Size exclusion chromatography to detect intact Y complex (~575 kDa)

• Sucrose gradient fractionation to separate assembled NPCs from soluble components

• Proximity labeling techniques (BioID, APEX) to map interaction networks

Microscopy-based methods:

• Super-resolution microscopy (SIM/STORM) to visualize NPC distribution

• Quantify co-localization with other NPC markers like:

  • Scaffold: Nup107, Nup96, Nup85

  • Basket: Nup153, Tpr

  • Central channel: Nup62

• Measure Nup153/Nup98 ratio as indicator of proper assembly

Functional assays:

• Nuclear transport assays using fluorescent import/export cargo

• mRNA export activity measurement

• Live-cell imaging with GFP-Nup133 to monitor dynamics

• FRAP to measure stability of incorporation

Research data shows that in Nup133−/− mESCs:

• NPCs form but approximately half fail to assemble/maintain a Tpr-containing nuclear basket

• The number of Tpr-stained NPCs is reduced by ~50% compared to wild-type cells

• GFP-Nup153 shows increased dynamics and decreased affinity for NPCs, indicating altered stability

• NPC numbers increase ~2-fold between G1 and G2 phases despite Nup133 absence

How can researchers investigate the specific role of NUP133 in RNA export independent of its structural role in the NPC?

Dissecting NUP133's specific role in RNA export requires approaches that separate its structural and functional contributions:

Domain-specific mutation strategies:

• Target domains outside the Nup84 interaction region

• Create point mutations that disrupt RNA binding but maintain structural integrity

• Generate ALPS motif mutations (e.g., replacing residues 252-270 with GGGGSGGGS) to affect membrane association while preserving protein-protein interactions

RNA transport assays:

• Oligo(dT) FISH to visualize poly(A)+ RNA distribution

• MS2-tagged RNA tracking for specific transcript export

• EU (5-ethynyl uridine) pulse-chase to monitor newly synthesized RNA

• Single-molecule RNA imaging to track export kinetics

Rescue experiment design:

• Express wild-type NUP133 vs. domain mutants in Nup133−/− cells

• Quantify restoration of:

  • NPC structure (by immunofluorescence)

  • RNA export (by FISH)

  • Cell growth and division

  • Interaction with Y complex partners

Inducible depletion systems:

• Auxin-inducible degron (AID) tagging for rapid NUP133 removal

• Conditional knockout systems

• siRNA with rescue using siRNA-resistant constructs

Data from research indicates that NUP133 is essential for RNA export as part of the Nup160 nuclear pore subcomplex (which includes Nup160, Nup96, and Nup107), ensuring mRNA and other RNA species are efficiently transported from nucleus to cytoplasm for translation . The specific mechanism appears to involve providing structural support for other nucleoporins directly involved in RNA binding and transport.

What experimental designs can elucidate the interaction between NUP133 and Cenp-F during cell division?

The interaction between NUP133 and Cenp-F represents a crucial link between the nuclear pore and mitotic processes. Researchers can investigate this relationship through:

Interaction mapping methods:

• Yeast two-hybrid assays with domain deletions/mutations

• In vitro binding assays with recombinant proteins

• In silico structural modeling to predict interaction interfaces

• Site-directed mutagenesis targeting specific residues:

  • V89D/M92D/T96D mutations in Nup133 α1 helix

  • L2681E/L2683E mutations in Cenp-F

Live cell imaging approaches:

• Dual-color imaging of fluorescently tagged NUP133 and Cenp-F

• FRET/BRET to detect direct interactions

• Temporal analysis of co-localization during mitotic progression

• Photoactivation studies to track protein movement

Functional perturbation experiments:

• Expression of dominant-negative mutants:

  • Nup133 α1 helix mutants (V89D/M92D/T96D)

  • Cenp-F miniSID mutants (L2681E/L2683E)

• Analysis of mitotic progression, chromosome alignment, and segregation

• Combined depletion experiments to test redundancy vs. synergy

Research findings demonstrate that:

• NUP133 interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis

• The interaction involves a conserved helix within the Nup133 β-propeller and a short leucine zipper-containing dimeric segment of Cenp-F

• Point mutations affecting the Nup133/Cenp-F interface prevent Cenp-F localization to the nuclear envelope but not to kinetochores

• This suggests separable functions for the interaction at different cell cycle stages

What are the most common technical challenges when using NUP133 antibodies and how can they be addressed?

Researchers frequently encounter specific technical challenges when working with NUP133 antibodies:

High background in immunofluorescence:

• Increase blocking time (≥1 hour) with 5% BSA or normal serum

• Optimize antibody dilution (test range from 1:100-1:1000)

• Include 0.1% Tween-20 in wash buffers

• Consider specialized fixation methods (e.g., pre-extraction with detergent before fixation)

• Use monoclonal antibodies for higher specificity when available

Multiple bands in Western blot:

• Expected molecular weight is 129-133 kDa

• Lower bands may represent degradation products

• Higher bands may indicate post-translational modifications

• Use fresh lysates with protease inhibitors

• Include phosphatase inhibitors to maintain modification state

• Consider subcellular fractionation to enrich nuclear membrane components

Weak or no signal in certain applications:

• For IP: Increase antibody amount (10 μg/mg lysate recommended)

• For Western blot: Try membrane stripping and re-probing methods

• For challenging applications, use antibodies specifically validated for that technique

• Consider epitope retrieval methods for fixed tissues

• Test different antibody clones targeting different epitopes

Species cross-reactivity issues:

• Nup133 shows conservation across species, but epitope differences exist

• For non-standard model organisms, check sequence homology at antibody epitope

• Consider using antibodies raised against conserved regions

• Validate each new species with appropriate controls

Experimental design recommendations:

• Include positive control samples (A375, LO2, HeLa, NCI-H460 cell lysates)

• Use Nup133−/− mESCs as negative controls when available

• For quantitative applications, establish standard curves with recombinant proteins

• Consider fixation timing carefully as NUP133 localization changes during cell cycle

How can researchers differentiate between technical artifacts and genuine biological findings when studying NUP133 distribution patterns?

Distinguishing artifacts from true biological patterns requires systematic validation:

Control experiments to implement:

• Side-by-side comparison of multiple antibody clones

• Secondary antibody-only controls to assess background

• Pre-adsorption controls using recombinant NUP133

• Genetic validation using siRNA knockdown or CRISPR knockout

• Rescue experiments with fluorescently tagged NUP133

Pattern validation approaches:

• Co-localization with other NPC components:

  • Scaffold: Nup107, Nup96 (should show high overlap)

  • Basket: Tpr, Nup153 (should show partial overlap)

• Quantitative assessment of co-localization coefficients

• Super-resolution microscopy to resolve substructures

• Electron microscopy correlation for ultrastructural confirmation

Cell cycle-specific considerations:

• Synchronize cells to specific cell cycle stages

• Compare patterns across G1, S, G2, and mitotic phases

• Remember that during mitosis, NUP133 legitimately redistributes to kinetochores

• In telophase, NUP133 reassociates with reforming nuclear envelope at chromosome-attached regions

Distinguishing features of authentic patterns:

• Nuclear rim staining with punctate distribution in interphase

• ~2-fold increase in NPC number between G1 and G2 phases (2.8 ± 0.9 × 10³ NPCs in G2)

• Approximately 50% of NPCs should be positive for both NUP133 and Tpr

• Heterogeneity in nuclear basket composition is a genuine biological finding, not an artifact

Research data shows that in WT mESCs, the number of Tpr-labeled NPCs doubles between G1 and G2 in both WT and Nup133−/− mESCs, indicating that observed changes in NPC composition across the cell cycle represent true biological variation rather than technical artifacts .

How can NUP133 antibodies be leveraged for studying nuclear pore complex assembly mechanisms?

NUP133 antibodies provide powerful tools for investigating NPC assembly through several sophisticated approaches:

Live-cell imaging strategies:

• Create NUP133 antibody-coated beads that capture GFP-NUP133 in living cells

• Live-CLEM imaging to visualize membrane fenestrations around beads

• Pulse-chase experiments with photoactivatable NUP133 to track new assembly

• FRAP studies to measure incorporation rates into forming NPCs

Assembly intermediates analysis:

• Extract timing information through cell synchronization experiments

• Characterize salt-extractable versus resistant fractions during assembly

• Immunoprecipitate NUP133 at different assembly stages to capture changing interaction partners

• Compare interphase versus post-mitotic assembly mechanisms

Structural analysis approaches:

• Use antibodies as fiducial markers for electron tomography

• Apply proximity-dependent labeling (BioID/TurboID) with NUP133 as bait

• Correlate with super-resolution microscopy data

• Nanobody-based approaches for structural studies

Research findings show significant insights:

• NUP133 can assemble NPC-like structures as the sole effector molecule on membrane fenestrations

• NUP133-coated beads effectively assemble Nup107 and ELYS, while showing minimal assembly of Nup98 and Nup62

• In metaphase, fenestrations on the ER membrane are observed around chromosomes

• In telophase, these fenestrations become filled at chromosome-attached regions, where NPC assembly occurs

• The ALPS motif of NUP133 NTD produces small fringe-like protrusions on liposome surfaces

What emerging technologies and methodologies are enhancing NUP133 antibody applications in current research?

Cutting-edge technologies are revolutionizing NUP133 research:

Advanced imaging techniques:

• Super-resolution microscopy beyond the diffraction limit:

  • Structured Illumination Microscopy (SIM)

  • Stochastic Optical Reconstruction Microscopy (STORM)

  • Stimulated Emission Depletion (STED)

• Expansion microscopy for physical magnification of specimens

• Lattice light-sheet microscopy for rapid 3D imaging with reduced phototoxicity

• Cryo-electron tomography for near-atomic resolution of NPC components

Novel antibody engineering approaches:

• Nanobodies (VHH-SAN4, 5, 8, and 9) for structural studies and live imaging

• Bi-specific antibodies targeting NUP133 and interaction partners

• Split-fluorescent protein complementation for direct visualization of interactions

• Intrabodies for tracking NUP133 in living cells

Integrative structural biology methods:

• Combining nanobody-bound crystal structures with cryo-EM maps

• Molecular dynamics simulations informed by antibody epitope mapping

• Cross-linking mass spectrometry with antibody-based pulldowns

• Hydrogen-deuterium exchange mass spectrometry for dynamics studies

Functional genomics integration:

• CRISPR screens with antibody-based readouts

• Optogenetic control of NUP133 coupled with antibody detection

• Single-cell profiling of NPC composition heterogeneity

• Spatial transcriptomics correlation with NUP133 distribution

Research examples demonstrate that:

• Nanobody-bound structures have enabled complete structural description of the entire 575 kDa Y complex from S. cerevisiae

• Live CLEM imaging with NUP133 antibodies reveals dynamics of ER membrane fenestrations during NPC assembly

• Structure of Nup84-Nup133 CTD details the high flexibility of this dimeric unit of the Y complex

• These technologies provide unprecedented insights into NPC structure, assembly, and function

How can NUP133 antibodies be utilized in research investigating disease associations?

NUP133 has been implicated in several diseases, and antibodies offer critical tools for investigating these connections:

Nephrotic syndrome research applications:

• Tissue expression profiling in patient biopsies

• Quantification of NUP133 levels in disease versus healthy tissues

• Co-immunoprecipitation studies to identify altered interaction networks

• Subcellular localization studies in disease models

Cancer research approaches:

• Analysis of NUP133 expression in tumor versus normal tissue microarrays

• Correlation with nuclear transport alterations in cancer progression

• Evaluation as potential diagnostic/prognostic marker

• Target validation for therapies disrupting nuclear transport

Viral infection studies:

• Tracking NUP133 redistribution during viral hijacking of nuclear transport

• Interaction studies with viral nuclear transport factors

• Monitoring changes in NPC composition during infection

• Differential display of NUP133 epitopes during infection

Methodology considerations:

• Patient-derived samples require optimized fixation protocols

• Quantitative analysis methods should include:

  • Digital pathology approaches

  • Automated image analysis for unbiased quantification

  • Correlation with clinical metadata

• Controls should include both healthy tissue and disease-relevant controls

Research data indicates that NUP133 is associated with nephrotic syndrome , suggesting altered nuclear transport may contribute to disease pathology. The protein's role in RNA export and macromolecular transport points to potential mechanisms in diseases involving dysregulated gene expression or protein mislocalization.

What considerations are important when designing experiments to investigate NUP133 mutations or alterations in disease models?

Investigating NUP133 alterations in disease contexts requires careful experimental design:

Mutation analysis strategies:

• Distinguish between:

  • Loss-of-function mutations (structural/stability impact)

  • Altered-function mutations (specific domain impacts)

  • Expression level changes (transcriptional/post-transcriptional)

• Design domain-specific antibodies to detect truncated variants

• Consider post-translational modification-specific antibodies

Model system selection:

• Patient-derived primary cells when available

• CRISPR-engineered cell lines with specific mutations

• Animal models with equivalent mutations

• iPSC-derived specialized cell types for tissue-specific effects

Functional readouts to consider:

• Nuclear transport assays (import/export dynamics)

• RNA-seq to detect transcriptome-wide effects

• Interactome changes via IP-mass spectrometry

• Nuclear envelope ultrastructure via electron microscopy

• NPC density and distribution quantification

Controls and validation approaches:

• Include wild-type rescue experiments

• Use multiple antibody clones targeting different epitopes

• Include domain-specific rescue constructs

• Correlate with patient phenotype data when possible

Research findings on NUP133 mutations show:

• Deletions of both complete NUP133 and portions of its N-terminus result in decreased fitness in yeast and clustering of NPCs on the nuclear envelope

• In humans, deletion of just the DA 34 loop (containing the ALPS motif) is essential for interphase assembly

• Nup133−/− mESCs show heterogeneity in nuclear basket composition, with approximately one-half of NPCs failing to assemble/maintain a Tpr-containing nuclear basket

• These findings suggest precise mutations can have distinct phenotypic consequences depending on the affected domain