PSMB4 Antibody

What is PSMB4 and why is it important in research?

PSMB4 is a β4 subunit of the 20S proteasome and a member of the ubiquitin-proteasome family. It plays a crucial role in the cytoplasmic protein catabolic process through the proteasome complex, which is an ATP-dependent multi-catalytic proteinase complex essential for degradation of intracellular targeted proteins . PSMB4 has gained significant research attention due to its involvement in multiple cancer types including breast cancer, glioblastoma, hepatocellular carcinoma, and pulmonary neuroendocrine tumors . Additionally, PSMB4 has been shown to possess antiviral properties against viruses such as PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) .

What cell lines and tissues express detectable levels of PSMB4?

PSMB4 is widely expressed across multiple human and mouse tissues and cell lines. Based on experimental validation, the following samples have shown positive PSMB4 expression:

What are the recommended applications and dilutions for PSMB4 antibodies?

PSMB4 antibodies have been validated for multiple experimental applications with specific optimized dilutions:

For optimal results in Western blotting, mouse anti-PSMB4 (clone OTI5B5) has been specifically validated at 1/1000 dilution for detecting PSMB4 in Jurkat cell lysates .

How should PSMB4 antibody samples be prepared and stored?

PSMB4 antibodies are typically supplied as purified IgG in liquid form. For instance, mouse anti-PSMB4 (clone OTI5B5) is prepared by affinity chromatography from ascites and formulated in phosphate buffered saline with preservative stabilizers (0.09% Sodium Azide, 1% Bovine Serum Albumin, and 50% Glycerol) . For optimal performance:

• Store the antibody at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles which can damage antibody integrity

• When working with the antibody, keep it on ice

• Most manufacturers guarantee stability for 12 months from the date of dispatch when stored properly

• For diluted working solutions, prepare fresh on the day of use

How can PSMB4 antibodies be used to investigate cancer progression mechanisms?

PSMB4 has been implicated in the progression of several cancer types, making its detection crucial for oncology research. Advanced approaches include:

• Proliferation and viability analysis: PSMB4 overexpression has been linked to enhanced cell growth and viability in breast cancer cells, contributing to poor prognosis . Researchers can use PSMB4 antibodies in combination with proliferation markers (e.g., Ki-67) to assess correlation between PSMB4 expression and cellular proliferation.

• Invasion and migration assessment: Studies in glioblastoma have shown that PSMB4 inhibition decreases proliferation, migration, and invasion abilities. After PSMB4 knockdown, researchers can use antibodies to monitor changes in invasion-related proteins, including phosphorylated focal adhesion kinase and matrix metallopeptidase 9 .

• Cell cycle and apoptosis investigation: Downregulated PSMB4 has been shown to result in cell cycle arrest and apoptosis in vitro . Researchers can employ flow cytometric analysis with Annexin V and propidium iodide staining after manipulating PSMB4 expression to monitor apoptotic changes.

• Therapeutic target validation: In orthotopic xenograft mouse models, decreased PSMB4 expression enhanced the anti-tumor effect of temozolomide (TMZ) on glioblastoma growth . PSMB4 antibodies can be used to confirm knockdown efficiency in such combination therapy studies.

What protocols enable optimal detection of PSMB4 in different cancer tissues?

For robust detection of PSMB4 across various cancer tissues, researchers should consider these optimized protocols:

• For pulmonary neuroendocrine tumors: PSMB4 shows differential expression between tumor subtypes, with the highest expression and greatest range in Large Cell Neuroendocrine Carcinomas (LCNEC) . For such heterogeneous tissues:

  • Use both mRNA analysis (TaqMan qPCR) and protein detection (IHC)

  • Compare results across different tumor subtypes (TC, AC, SCLC, LCNEC)

  • Correlate PSMB4 expression with proliferation markers like Ki-67

• For breast cancer tissues:

  • Perform antigen retrieval with TE buffer at pH 9.0

  • If background staining occurs, alternative antigen retrieval with citrate buffer (pH 6.0) may be used

  • Include normal breast tissue controls

  • Use positive controls known to express PSMB4 (e.g., MCF-7 cells)

• For glioblastoma tissues:

  • Correlate PSMB4 antibody staining with patient survival data

  • Consider using tissue microarrays (TMAs) for high-throughput analysis

  • Include analysis of associated signaling pathways (e.g., NF-κB activity)

How can researchers investigate the relationship between PSMB4 expression and proteasome inhibitor efficacy?

Proteasome inhibitors provide a unique approach to cancer therapy by depressing proteasome function. As PSMB4 is a key proteasomal subunit, studying its relationship to inhibitor efficacy is valuable:

• Correlation studies: Use PSMB4 antibodies to quantify expression levels in patient-derived samples before treatment with proteasome inhibitors like carfilzomib or bortezomib .

• Resistance mechanisms: In treatment-resistant cell lines, monitor changes in PSMB4 expression using Western blot to determine if altered expression correlates with decreased sensitivity.

• Combination therapy assessment: As demonstrated in glioblastoma, where PSMB4 downregulation enhanced temozolomide efficacy , researchers can use PSMB4 antibodies to:

  • Confirm knockdown efficiency in preclinical models

  • Monitor PSMB4 expression changes during treatment

  • Correlate expression levels with therapeutic outcomes

• Proteasome activity correlation: Use PSMB4 antibodies in parallel with proteasome activity assays to determine whether expression levels directly correlate with functional changes after inhibitor treatment.

What approaches allow researchers to study PSMB4's role in antiviral mechanisms?

Recent research has revealed that PSMB4 plays an antiviral role against viruses such as PRRSV . To investigate this function:

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with PSMB4 antibodies to identify viral protein interactions

  • GST-pulldown assays can confirm direct interactions

  • For PRRSV specifically, investigate interactions with Nsp1α using PSMB4 antibodies

• Ubiquitination analysis:

  • PSMB4 mediates K63-linked ubiquitination of PRRSV Nsp1α at K169

  • Use PSMB4 antibodies alongside ubiquitin-specific antibodies in Western blot analysis

  • Perform immunofluorescence to visualize co-localization of PSMB4, ubiquitin, and viral proteins

• Degradation pathway investigation:

  • PSMB4 triggers viral protein degradation via the lysosomal pathway by interacting with LC3

  • Use PSMB4 antibodies in conjunction with lysosomal markers

  • Monitor autophagosome formation with confocal microscopy

• Signaling pathway analysis:

  • PSMB4 activates the NF-κB signaling pathway to produce type I interferons

  • Use PSMB4 antibodies alongside antibodies against IκBα, p-IκBα, and interferon signaling components

What are the common technical challenges when using PSMB4 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with PSMB4 antibodies:

• Non-specific binding in Western blots:

  • Increase blocking time (use 5% non-fat milk or BSA in TBST for 1-2 hours)

  • Optimize antibody dilution (start with 1:2000 and adjust as needed)

  • Include additional washing steps with TBST

  • Consider using different secondary antibodies

• Weak signals in IHC:

  • Ensure proper antigen retrieval (TE buffer pH 9.0 is recommended for PSMB4)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification systems like biotin-streptavidin

  • Reduce background with appropriate blocking (use 10% normal serum from the species of secondary antibody)

• Variable results across different samples:

  • Standardize protein extraction methods

  • Normalize loading in Western blots using housekeeping proteins

  • Process all samples simultaneously under identical conditions

  • Include positive controls (e.g., Jurkat cells for Western blot)

How can PSMB4 antibodies be validated for specificity and reproducibility?

Thorough validation ensures reliable research results:

• Knockdown/knockout validation:

  • Perform siRNA knockdown of PSMB4 in relevant cell lines

  • Compare antibody signal between control and PSMB4-depleted samples

  • The 28 kDa band should be significantly reduced in knockdown samples

• Multiple antibody comparison:

  • Use antibodies from different suppliers or clones

  • Verify consistent staining patterns across antibodies

  • Compare monoclonal versus polyclonal antibodies for confirmation

• Multiple application verification:

  • Confirm PSMB4 detection across different techniques (WB, IHC, IF)

  • Ensure consistent results between protein and mRNA expression analysis

  • For example, verify if PSMB4 protein levels detected by IHC correlate with mRNA levels measured by qPCR, as observed in pulmonary neuroendocrine tumors

• Cross-species reactivity testing:

  • If working with different species, verify specificity in each relevant model

  • Compare sequence homology and potential epitope conservation

How can PSMB4 antibodies contribute to understanding potential therapeutic resistance mechanisms?

As PSMB4 overexpression is associated with cancer progression, investigating its role in therapeutic resistance is valuable:

• Monitoring expression changes during treatment:

  • Use PSMB4 antibodies to quantify expression before and after treatment

  • Correlate changes with clinical outcomes

  • Determine if PSMB4 upregulation occurs in response to therapy pressure

• Investigating compensatory mechanisms:

  • After proteasome inhibitor treatment, use PSMB4 antibodies to evaluate if compensatory upregulation occurs

  • Assess potential structural changes or post-translational modifications in PSMB4 that could alter inhibitor binding

• Combination therapy development:

  • Use PSMB4 antibodies to evaluate the effects of novel drug combinations

  • As demonstrated in glioblastoma, where PSMB4 downregulation enhanced temozolomide efficacy , similar approaches could be explored in other cancers

What are advanced approaches for studying PSMB4's role in the tumor microenvironment?

Beyond cancer cell studies, PSMB4's role in the tumor microenvironment warrants investigation:

• Multiplex immunofluorescence:

  • Use PSMB4 antibodies in combination with markers for different cell types (T cells, macrophages, fibroblasts)

  • Evaluate spatial relationships between PSMB4-expressing cells and other components of the tumor microenvironment

• Single-cell analysis:

  • Combine PSMB4 antibody staining with single-cell sorting

  • Perform downstream transcriptome or proteome analysis on PSMB4-high versus PSMB4-low cells

• 3D organoid models:

  • Use PSMB4 antibodies to monitor expression changes in 3D cultures

  • Compare expression patterns between 2D and 3D environments

  • Evaluate how stromal interactions affect PSMB4 expression in cancer cells