wsb1 Antibody

What is WSB1 and why is it significant in research?

WSB1 (WD repeat and SOCS box-containing protein 1) functions as a substrate recognition subunit of the ECS (Elongin B/C–Cul2/5–SOCS) ubiquitin ligase complexes. It contains seven WD40 repeats spanning most of the protein and a SOCS box in the C-terminus. Its significance stems from its role in various cellular processes, particularly as an E3 ubiquitin ligase that targets proteins for degradation. WSB1 has been implicated in cancer development through its regulation of key proteins such as ATM and pVHL, making it an important research target .

What are the molecular characteristics of WSB1 that researchers should be aware of?

WSB1 has a calculated molecular weight of 47 kDa (421 amino acids), though it typically appears at approximately 56 kDa in experimental conditions. It contains multiple functional domains: several WD-repeats that facilitate protein-protein interactions and a SOCS box domain that enables formation of E3 ubiquitin ligase complexes. The gene has alternatively spliced transcript variants encoding three distinct isoforms. WSB1 undergoes post-translational modifications, particularly CDK-mediated phosphorylation, which activates WSB1 by promoting its monomerization .

How do I select the appropriate WSB1 antibody for my research?

Selection criteria should include:

• Target specificity: Confirm reactivity with your species of interest (human, mouse, etc.)

• Application compatibility: Verify validation for your intended application (WB, IHC, IF/ICC)

• Isotype and host: Consider rabbit polyclonal options for broader epitope recognition

• Published validation: Review antibodies used in peer-reviewed research

• Recognition domain: Consider whether specific domains or isoforms need targeting

For example, antibody 11666-1-AP has been validated for WB, IHC, and IF/ICC applications with human samples and has been cited in multiple publications .

What are the recommended protocols for using WSB1 antibody in Western blotting?

For optimal Western blot results with WSB1 antibody:

• Sample preparation: Extract proteins from cells (e.g., COLO 320, HepG2, SMMC-7721) using standard lysis buffers containing protease inhibitors

• Protein loading: Load 20-40 μg of total protein per lane

• Separation: Use 10-12% SDS-PAGE gels for optimal resolution around the 56 kDa mark

• Transfer: Standard PVDF or nitrocellulose membranes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute WSB1 antibody 1:1000-1:6000 in blocking buffer

• Incubation: Overnight at 4°C with gentle agitation

• Detection: Use appropriate secondary antibody and detection system

• Expected band: ~56 kDa (observed molecular weight)

Note that experimental optimization may be required based on your specific cell lines or tissues .

How should researchers optimize immunohistochemistry protocols for WSB1 detection?

For specific cancer research applications, particularly in prostate cancer, the antibody has been successfully employed with these parameters. Always include positive and negative controls to validate staining specificity .

What methodological considerations are important for immunofluorescence studies with WSB1 antibody?

For successful IF/ICC experiments:

• Cell preparation: Culture cells (HepG2 cells recommended as positive control) on appropriate coverslips

• Fixation options: 4% paraformaldehyde (10-15 minutes at room temperature) or ice-cold methanol (10 minutes)

• Permeabilization: 0.1-0.3% Triton X-100 in PBS (if using paraformaldehyde fixation)

• Blocking: 1-5% BSA or normal serum in PBS for 30-60 minutes

• Primary antibody: Dilute WSB1 antibody 1:50-1:500 in blocking solution

• Incubation: 1-2 hours at room temperature or overnight at 4°C

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG at manufacturer's recommended dilution

• Counterstaining: DAPI for nuclei visualization

• Mounting: Anti-fade mounting medium

Note that WSB1 localization may vary depending on cellular context and experimental conditions, so appropriate controls should be included .

How can researchers study the E3 ligase activity of WSB1 in cancer progression models?

To investigate WSB1's E3 ligase function:

• In vitro ubiquitination assays:

  • Purify recombinant WSB1 protein (full-length and ΔSOCS mutant as negative control)

  • Combine with E1, E2 enzymes, ubiquitin, ATP, and substrate protein (ATM or pVHL)

  • Analyze ubiquitination by Western blot with anti-ubiquitin antibody

• Cell-based degradation assays:

  • Manipulate WSB1 expression through overexpression or siRNA knockdown

  • Monitor substrate protein levels (pVHL or ATM) by Western blot

  • Include proteasome inhibitor (MG132) controls to confirm degradation pathway

  • Assess protein half-life through cycloheximide chase experiments

• Substrate specificity analysis:

  • Perform co-immunoprecipitation to confirm direct interaction with substrates

  • Create deletion mutants to map interaction domains

  • Utilize the ΔSOCS box mutant as a negative control for ligase activity

Published work demonstrates that WSB1 promotes ATM and pVHL ubiquitination, resulting in their degradation, which can be reversed by proteasome inhibitors .

What experimental approaches should be used to investigate the relationship between WSB1 and HIF signaling?

To study WSB1-HIF signaling interactions:

• Hypoxia response element (HRE) reporter assays:

  • Transfect cells with HRE-luciferase reporter

  • Manipulate WSB1 expression and assess HIF transcriptional activity

  • Compare normoxic vs. hypoxic conditions

• Gene expression analysis:

  • Measure HIF target genes (VEGFA, ALDOC, CA9, SAP30) by qRT-PCR after WSB1 manipulation

  • Perform RNA-seq to identify broader transcriptional changes

  • Validate findings with ChIP assays for HIF binding to target promoters

• Protein stability studies:

  • Examine pVHL, HIF-1α, and HIF-2α protein levels following WSB1 overexpression or knockdown

  • Compare effects in VHL-competent vs. VHL-deficient cell lines (e.g., RCC4, 786-O, and their VHL-reconstituted derivatives)

  • Perform cycloheximide chase experiments to assess protein stability

• Feedback loop investigation:

  • Silence HIF-1α and examine WSB1 expression under hypoxia

  • Use ChIP to confirm HIF-1 binding to the WSB1 promoter

  • Create reporter constructs with the WSB1 promoter to validate direct regulation

Research demonstrates that WSB1 and HIF-1 form a positive feedback loop, with HIF-1 inducing WSB1 expression, and WSB1 stabilizing HIF-1α by promoting pVHL degradation .

How can researchers differentiate between WSB1's roles in cellular transformation versus metastatic progression?

To distinguish these distinct oncogenic functions:

• Cellular transformation models:

  • Use primary cells (not pre-transformed lines) for transformation assays

  • Assess OIS bypass through proliferation after oncogene introduction

  • Measure senescence markers (SA-β-gal, p16, p21) and ATM pathway components

  • Perform soft agar colony formation assays to quantify anchorage-independent growth

  • Monitor DNA damage response pathway activation (γH2AX foci)

• Metastasis models:

  • Conduct invasion and migration assays (Boyden chamber, wound healing)

  • Assess matrix metalloproteinase activity and epithelial-mesenchymal transition markers

  • Perform in vivo metastasis assays in appropriate animal models

  • Compare the expression of HIF target genes involved in metastasis

  • Use both VHL-competent and VHL-deficient cell lines to establish dependency

• Comparative analysis:

  • Generate WSB1 mutants selectively deficient in ATM or pVHL interaction

  • Assess which mutants rescue which phenotypes

  • Perform temporal studies to determine when each pathway is most active

Research indicates WSB1 promotes early tumorigenesis through ATM degradation (OIS bypass) and later metastatic progression through pVHL degradation and HIF stabilization .

How should researchers address inconsistent WSB1 antibody staining patterns?

When encountering inconsistent WSB1 staining:

• Antibody validation:

  • Confirm antibody specificity using WSB1 knockout or knockdown controls

  • Test multiple antibodies recognizing different epitopes

  • Verify antibody lot consistency and storage conditions

• Technical optimization:

  • Systematically test different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Titrate antibody concentration more precisely (1:50, 1:100, 1:200, 1:500)

  • Adjust incubation times and temperatures

  • Test different detection systems

• Biological considerations:

  • WSB1 expression varies across tissues and can be induced by hypoxia

  • Consider cell-type specific expression patterns

  • Evaluate subcellular localization which may change based on activation state

  • Account for potential isoform-specific expression

• Controls and validation:

  • Include known positive control tissues (human prostate cancer tissue)

  • Use cell lines with confirmed WSB1 expression (HepG2, COLO 320, SMMC-7721)

  • Consider complementary detection methods (IF with WB validation)

Inconsistent results may reflect genuine biological variation rather than technical issues, as WSB1 levels respond dynamically to hypoxia and other cellular conditions .

How can researchers reconcile discrepancies in observed molecular weight of WSB1 in Western blot experiments?

To address molecular weight variations:

• Expected values:

  • Calculated molecular weight: 47 kDa (421 amino acids)

  • Observed molecular weight: ~56 kDa (as reported in literature)

• Sources of variation:

  • Post-translational modifications (particularly phosphorylation by CDKs)

  • Alternative splicing (three known isoforms)

  • Sample preparation methods (denaturing conditions)

  • Gel percentage and running conditions

  • Presence of fusion tags in recombinant proteins

• Validation approaches:

  • Run positive control lysates alongside experimental samples

  • Include recombinant WSB1 protein as size reference

  • Perform knockdown/knockout controls to confirm band specificity

  • Use multiple antibodies targeting different epitopes

  • Perform phosphatase treatment to assess contribution of phosphorylation

• Documentation practices:

  • Report both predicted and observed molecular weights

  • Document gel percentage and running conditions

  • Note any treatments affecting post-translational modifications

The difference between calculated (47 kDa) and observed (56 kDa) molecular weights is consistent across multiple studies and likely reflects post-translational modifications or structural properties affecting migration .

How does WSB1's role in oncogene-induced senescence inform experimental design for cancer studies?

WSB1's role in OIS bypass has significant implications for experimental design:

• Cell model selection:

  • Use primary cells for senescence studies rather than immortalized lines

  • Consider models expressing oncogenes that trigger senescence (Ras, Myc)

  • Include ATM pathway components in analysis

• Experimental sequence:

  • Establish baseline senescence response to oncogene expression

  • Manipulate WSB1 levels before or after oncogene introduction

  • Monitor cell proliferation, senescence markers, and DNA damage

  • Track ATM levels and activation status (phospho-ATM)

• Mechanistic validation:

  • Rescue experiments with ATM overexpression

  • Use WSB1 mutants lacking E3 ligase activity (ΔSOCS)

  • Assess CDK-mediated phosphorylation of WSB1

  • Monitor ubiquitination status of ATM

• Translational relevance:

  • Examine WSB1 and ATM levels in premalignant versus malignant tissues

  • Correlate with proliferation and senescence markers

  • Consider potential for therapeutic targeting of WSB1-ATM axis

Research demonstrates that WSB1 overcomes OIS through targeting ATM for degradation, representing an early event in tumorigenesis that could be targeted therapeutically .

What methodological approaches are most effective for studying the WSB1-HIF-pVHL regulatory axis in metastatic progression?

For investigating this regulatory axis:

• Cell line selection:

  • Use paired VHL-deficient and VHL-reconstituted cell lines (RCC4/RCC4-VHL, 786-O/786-O-VHL)

  • Include cell lines with differential metastatic potential

  • Consider hypoxia-responsive versus constitutively HIF-active models

• Functional assays:

  • In vitro migration and invasion assays

  • 3D organoid invasion models

  • In vivo metastasis models with bioluminescent tracking

  • Endothelial tube formation and angiogenesis assays

• Molecular analysis:

  • Assess WSB1-pVHL-HIF-1α protein relationship through co-immunoprecipitation

  • Measure pVHL ubiquitination following WSB1 manipulation

  • Monitor HIF target gene expression (VEGFA, ALDOC, CA9, SAP30)

  • Compare normoxic versus hypoxic conditions

• Clinical correlation:

  • Analyze WSB1, pVHL, and HIF-1α levels in primary versus metastatic samples

  • Correlate WSB1 expression with metastasis-free survival

  • Stratify analysis by cancer subtypes (particularly important in breast cancer)

Research indicates WSB1 promotes metastasis through pVHL degradation, resulting in HIF stabilization even under normoxic conditions, with particularly strong effects in certain cancer subtypes .

How should researchers design experiments to evaluate potential therapeutic targeting of WSB1?

For therapeutic targeting investigations:

• Target validation approaches:

  • CRISPR/Cas9 knockout versus transient knockdown comparisons

  • Rescue experiments with wild-type versus mutant WSB1

  • Assess phenotypic consequences in multiple cell types

  • Evaluate effects in 3D and in vivo models

• Inhibition strategies:

  • Develop assays for WSB1 E3 ligase activity amenable to screening

  • Target the SOCS box-elongin interaction

  • Block CDK-mediated activation of WSB1

  • Disrupt WSB1-substrate (ATM/pVHL) interactions

• Context-dependent efficacy:

  • Test in hypoxic versus normoxic conditions

  • Evaluate in VHL-deficient versus VHL-proficient backgrounds

  • Compare primary versus metastatic models

  • Assess efficacy in different cancer subtypes

• Biomarker development:

  • Correlate WSB1 activity with therapeutic response

  • Develop assays for WSB1 phosphorylation status

  • Monitor substrate levels (ATM, pVHL) as pharmacodynamic markers

  • Track HIF target gene expression as functional readout

Given WSB1's dual roles in tumor initiation (via ATM degradation) and metastatic progression (via pVHL degradation), therapeutic targeting could potentially address both early and late stages of cancer development .