CUL4B Antibody

What is CUL4B and what cellular functions does it regulate?

CUL4B (Cullin 4B) functions as a core component of multiple cullin-RING-based E3 ubiquitin-protein ligase complexes that mediate the ubiquitination and subsequent proteasomal degradation of target proteins . Within these complexes, CUL4B serves as a scaffold protein, contributing to catalysis by positioning both the substrate and the ubiquitin-conjugating enzyme .

CUL4B participates in numerous cellular processes including:

• Cell cycle regulation, particularly G1 progression through cyclin E ubiquitination

• DNA damage response via polyubiquitination of CDT1, histone H2A, histone H3, and histone H4

• Regulation of the mammalian target-of-rapamycin (mTOR) pathway controlling cell growth and metabolism

• Neural progenitor cell growth and mitosis progression

• T cell activation and expansion processes

The functional specificity of CUL4B-containing E3 ligase complexes depends on variable substrate recognition subunits that determine which proteins are targeted for degradation .

What different isoforms of CUL4B exist and how can they be distinguished?

Three major CUL4B isoforms have been identified in human and rodent cells :

• CUL4B-1: The canonical form comprising 913 amino acids (104 kDa)

• CUL4B-2: Missing the first 22 amino acids compared to CUL4B-1

• CUL4B-3: Missing the first 196 amino acids and differs in residues 197-203

These isoforms exhibit different neddylation patterns, with larger isoforms (CUL4B-1 and CUL4B-2) predominantly unneddylated despite possessing the C-terminal neddylation consensus site . The extended N-terminus in CUL4B-1 and CUL4B-2 appears to inhibit neddylation .

For experimental differentiation:

• Use high-resolution SDS-PAGE to separate the isoforms based on molecular weight

• Employ antibodies that recognize specific isoforms or regions

• Use anti-NEDD8 antibodies in conjunction with anti-CUL4B to distinguish neddylated forms

How should researchers validate CUL4B antibody specificity?

Rigorous validation of CUL4B antibodies is essential due to potential cross-reactivity with the highly homologous CUL4A (83% identity) . Recommended validation approaches include:

• Overexpression systems:

  • Transfect cells with GFP-tagged CUL4B fragments

  • Confirm antibody recognition of the tagged protein by immunoblotting

• Cross-reactivity testing:

  • Perform immunoprecipitation with the anti-CUL4B antibody

  • Probe precipitates with antibodies against related cullins (CUL4A, CUL1)

  • Verify that the antibody precipitates CUL4B but not other cullins

• Molecular weight verification:

  • Confirm antibody detection at the expected molecular weights for CUL4B isoforms

  • CUL4B-1: ~104 kDa; CUL4B-2: slightly smaller; CUL4B-3: smallest isoform

• Co-immunoprecipitation of known interactors:

  • Verify that the antibody co-precipitates established CUL4B binding partners like RBX1 and DDB1

Research has shown that some antibodies raised against N-terminal regions have reduced efficiency in recognizing CUL4B-3, which lacks these sequences .

What experimental applications are CUL4B antibodies suitable for?

CUL4B antibodies have been validated for multiple research applications:

• Western blotting:

  • Detection of endogenous CUL4B expression levels

  • Identification of different CUL4B isoforms

  • Analysis of post-translational modifications including neddylation

• Immunoprecipitation:

  • Isolation of CUL4B protein complexes

  • Co-immunoprecipitation studies to identify interacting partners

• Immunofluorescence microscopy:

  • Subcellular localization studies

  • Co-localization with mitotic markers or substrate proteins

• Flow cytometry and cytometric bead arrays:

  • Some antibodies are available as matched pairs for quantitative analysis

• Multiplex assays:

  • ELISAs and multiplex imaging applications

  • Recombinant monoclonal antibodies in conjugation-ready formats are particularly suitable

Selection of the appropriate antibody should consider the specific isoforms or modifications being targeted, as antibody recognition efficiency may vary across different CUL4B forms .

What are the optimal conditions for CUL4B antibody storage and handling?

To maintain antibody performance and stability:

• Store recombinant monoclonal CUL4B antibodies at -80°C for long-term preservation

• Aliquot antibodies to minimize freeze-thaw cycles

• Working dilutions can be stored at 4°C for short-term use

• Follow manufacturer-specific recommendations as formulations may vary

For example, Proteintech's recombinant monoclonal CUL4B antibody (85001-4-PBS) is supplied in PBS without BSA or azide at 1 mg/mL and should be stored at -80°C .

How can researchers distinguish between neddylated and unneddylated CUL4B in experimental samples?

Differentiating between neddylation states is crucial for understanding CUL4B's functional regulation. Methodological approaches include:

• Gel mobility shift analysis:

  • Neddylation causes a subtle upward mobility shift on SDS-PAGE

  • Run samples on 8-10% gels for optimal separation

  • Perform sequential probing with anti-CUL4B and anti-NEDD8 antibodies

• Dual immunodetection approach:

  • Immunoprecipitate with anti-CUL4B antibody

  • Divide the precipitate and probe parallel blots with anti-CUL4B and anti-NEDD8

  • Neddylated CUL4B will be detected by both antibodies

• Immunofluorescence co-localization:

  • Co-stain cells with anti-CUL4B and anti-NEDD8 antibodies from different species

  • Analyze co-localization using confocal microscopy

  • Research shows that in neural progenitor and NT-2 cells, most CUL4B does not co-localize with NEDD8, indicating predominant unneddylated status

Understanding neddylation status is particularly important as research indicates that unneddylated CUL4B isoforms specifically inhibit β-catenin degradation during mitosis, suggesting distinct functional roles for differentially modified forms .

What methodological approaches are optimal for studying CUL4B in neural progenitor cells?

To investigate CUL4B in neural progenitor cells (NPCs), consider these experimental strategies:

• Localization studies:

  • Perform immunofluorescence microscopy in brain sections focusing on the subventricular zone and subgranular zone where CUL4B-positive NPCs are enriched

  • Co-stain with mitotic markers such as MPM-2 to assess association with cell cycle phases

  • Analyze co-expression with β-catenin, which has been shown to be highly expressed in CUL4B-positive cells in vivo

• Functional manipulation:

  • Use shRNA vectors co-expressing GFP to track transfected cells while downregulating CUL4B

  • Assess effects on cell cycle distribution using flow cytometry with DNA content analysis

  • Investigate mitosis progression and potential G2/M arrest

• Biochemical analysis:

  • Fractionate cellular components to examine CUL4B distribution between nuclear and cytoplasmic compartments

  • Analyze isoform distribution and neddylation status in NPCs compared to differentiated cells

  • Perform co-immunoprecipitation to identify NPC-specific CUL4B interactors

• Developmental studies:

  • Track CUL4B expression during neurogenesis

  • Correlate with cell proliferation and differentiation markers

  • Research has shown that CUL4B is necessary for mitosis progression in NPCs and its disruption arrests cells in G2/M phase

How should researchers design experiments to investigate CUL4B's role in cell cycle regulation?

When studying CUL4B's function in cell cycle control, consider these methodological approaches:

• Cell synchronization strategies:

  • Synchronize cells at specific cycle phases using established methods (thymidine block, nocodazole, etc.)

  • Analyze CUL4B expression, localization, and modification status across cell cycle phases

  • Pay particular attention to mitosis, where unneddylated CUL4B accumulates

• CUL4B manipulation approaches:

  • Implement transient knockdown using validated shRNAs

  • For stable depletion, consider inducible systems as complete knockout may be lethal

  • Rescue experiments with different CUL4B isoforms can reveal isoform-specific functions

• Substrate degradation analysis:

  • Monitor levels of known CUL4B substrates including cyclin E and β-catenin

  • Perform cycloheximide chase assays to assess protein stability

  • Analyze how CUL4B depletion affects substrate levels during specific cell cycle phases

• Mechanistic investigations:

  • Examine how neddylation status changes during cell cycle progression

  • Research indicates unneddylated CUL4B specifically inhibits β-catenin degradation during mitosis

  • Use co-immunoprecipitation to identify cell cycle-specific CUL4B interactors

Noteworthy findings show that CUL4B downregulation arrests the cell cycle in G2/M phase in NPCs and human NT-2 cells, highlighting its critical role in mitosis progression .

What approaches can researchers use to investigate CUL4B's function in T cell biology?

To study CUL4B's role in T cell expansion and function:

• Activation models:

  • Isolate CD4+ T cells and activate with anti-CD3/CD28 antibodies

  • Monitor changes in CUL4B expression and neddylation following TCR stimulation

  • Research shows both total and neddylated CUL4B increase after TCR stimulation

• Functional assessment:

  • Measure proliferation using CFSE dilution or BrdU incorporation

  • Evaluate cell survival with Annexin V/PI staining

  • Studies demonstrate CUL4B disruption impairs both proliferation and survival of activated T cells

• Genetic manipulation:

  • Implement RNAi or CRISPR-Cas9 approaches to disrupt CUL4B expression

  • Design T cell-specific conditional knockout models for in vivo studies

  • Use retroviral expression systems for structure-function analyses with mutant forms

• Signaling pathway analysis:

  • Investigate how CUL4B affects TCR signaling cascade components

  • Identify T cell-specific CUL4B substrates using immunoprecipitation coupled with mass spectrometry

  • Examine changes in key transcription factors regulating T cell activation and differentiation

• In vivo relevance:

  • Perform adoptive transfer experiments comparing wild-type versus CUL4B-deficient T cells

  • Assess responses to infection or vaccination challenges

  • Investigate potential immunological disorders arising from CUL4B dysfunction

How can researchers differentiate between CUL4A and CUL4B functions in experimental systems?

Distinguishing between these highly homologous proteins (83% identity) requires careful experimental design:

• Isoform-specific knockdown:

  • Design siRNAs or shRNAs targeting unique regions

  • Validate knockdown specificity using antibodies that don't cross-react

  • Implement rescue experiments with RNAi-resistant constructs

• Comparative localization studies:

  • CUL4B accumulates in the nucleus during cell differentiation

  • Use cellular fractionation and immunofluorescence to compare subcellular distribution

  • Co-localization with specific substrates may indicate functional specificity

• Substrate specificity analysis:

  • Compare effects of CUL4A versus CUL4B depletion on potential substrates

  • CUL4B specifically downregulates WDR5 and Peroxiredoxin III

  • Analyze whether substrates are differentially affected by distinct CUL4B isoforms

• Structural and functional distinctions:

  • CUL4B contains an extended N-terminus absent in CUL4A

  • Investigate unique protein-protein interactions mediated by this region

  • Examine differential neddylation patterns between CUL4A and CUL4B

• Phenotypic analysis:

  • Compare cellular consequences of CUL4A versus CUL4B manipulation

  • CUL4B mutations specifically cause X-linked intellectual disability, suggesting unique neuronal functions

  • CUL4B is indispensable for embryonic development in mice

Understanding the distinct roles of these related proteins will provide insights into their non-redundant functions in cellular processes and disease mechanisms.