PSMC5 Antibody

What is PSMC5 and what cellular functions does it regulate?

PSMC5, also known as SUG1, RPT6, TRIP1, or p45, is a component of the 26S proteasome complex that functions in the degradation of ubiquitinated proteins . It belongs to the heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitinated target proteins for degradation . PSMC5 plays a key role in maintaining protein homeostasis by eliminating misfolded or damaged proteins that could impair cellular functions . This proteasome component participates in numerous cellular processes including cell cycle progression, apoptosis, and DNA damage repair . Recent research has identified PSMC5's involvement in cancer progression, immune cell regulation, and neuroinflammatory processes .

What applications are PSMC5 antibodies validated for?

PSMC5 antibodies have been validated for multiple research applications:

• Western blotting (WB): For detecting PSMC5 protein expression levels in cell and tissue lysates

• Enzyme-linked immunosorbent assay (ELISA): For quantitative measurement of PSMC5

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For examining PSMC5 expression patterns in tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing PSMC5 localization within cells

When selecting a PSMC5 antibody, researchers should verify its reactivity with their species of interest. The antibodies described in the search results have been validated for human samples, with some also confirmed for mouse and rat samples .

What experimental controls should be included when working with PSMC5 antibodies?

For rigorous PSMC5 antibody-based experiments, researchers should implement several controls:

• Positive controls: Use cell lines with known PSMC5 expression such as HepG2, MCF7, HeLa, 293T, or NIH/3T3

• Negative controls:

  • Primary antibody omission control

  • Isotype control (using matched IgG)

  • PSMC5 knockdown/knockout samples (using siRNA or CRISPR)

• Loading controls: For Western blotting, include housekeeping proteins (β-actin, GAPDH) to normalize protein loading

• Recombinant protein controls: When available, include purified PSMC5 protein as a reference standard

These controls help validate antibody specificity, minimize background signals, and ensure experimental reproducibility across different conditions.

How can researchers confirm PSMC5 antibody specificity?

Confirming antibody specificity is crucial for generating reliable research data. For PSMC5 antibodies, researchers can:

• Perform Western blotting to verify a single band at the expected molecular weight (~45 kDa)

• Use genetic approaches:

  • siRNA or shRNA knockdown of PSMC5 should reduce signal intensity proportionally to knockdown efficiency

  • CRISPR/Cas9 knockout of PSMC5 should eliminate specific binding

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide should block specific binding

• Cross-validate with multiple antibodies: Using antibodies targeting different epitopes of PSMC5 should yield consistent results

• Mass spectrometry validation: For advanced confirmation, immunoprecipitated proteins can be analyzed by mass spectrometry

Proper validation prevents misinterpretation of results due to potential cross-reactivity with other proteins.

How does PSMC5 contribute to cancer progression and immune modulation?

PSMC5 plays multifaceted roles in cancer progression, particularly in colorectal cancer (CRC), through several mechanisms:

• Proliferation and invasion: PSMC5 is significantly overexpressed in CRC tissues compared to normal tissues . Silencing PSMC5 dramatically suppresses proliferation and invasion of CRC cells, while overexpression enhances these properties .

• Epithelial-mesenchymal transition (EMT): PSMC5 activates EMT, a key process in cancer metastasis . Gene set enrichment analysis confirms PSMC5's correlation with EMT pathways .

• Immune cell infiltration regulation:

  • PSMC5 negatively correlates with CD8+ T cells and B cells (anti-tumor immune cells)

  • PSMC5 positively correlates with tumor-associated macrophages and neutrophils

  • PSMC5 is associated with higher expression of immune checkpoint molecules including PD-L1 (CD274), CTLA-4, and IDO1

• Chemokine regulation: PSMC5 positively correlates with CCL3, CCL4, and CCL5, which regulate tumor-associated macrophage and neutrophil abundance .

• Immune cell phenotype modulation: PSMC5 correlates with markers of protumorigenic M2 macrophages and N2 neutrophils, suggesting a role in promoting immunosuppressive phenotypes .

These findings indicate PSMC5 could be a promising biomarker and potential therapeutic target for immune therapy in CRC and possibly other cancers .

What techniques are most effective for studying PSMC5 knockdown effects?

For investigating PSMC5 function through knockdown approaches, researchers have successfully employed several techniques:

• RNA interference:

  • siRNA transfection: Effective for transient knockdown in cell lines. Studies have used this approach to demonstrate that silencing PSMC5 dramatically suppresses proliferation and invasion of colorectal cancer cells .

  • shRNA: For stable knockdown, shRNA targeting PSMC5 has been delivered via lentiviral vectors in both in vitro and in vivo studies . This approach has revealed PSMC5's role in LPS-induced cognitive deficits and neuroinflammation .

• CRISPR/Cas9 genome editing: For complete knockout studies, though this must be used carefully as PSMC5 is essential for cellular function.

• Validation of knockdown:

  • Western blotting to confirm protein reduction

  • qRT-PCR to confirm mRNA reduction

  • Functional assays to confirm biological effects

• Experimental readouts after PSMC5 knockdown:

  • Cell proliferation assays (MTT, colony formation)

  • Invasion and migration assays

  • Signaling pathway activation (Western blotting for phosphorylated proteins)

  • Gene expression profiling (RNA-seq)

  • Immune cell infiltration analysis (flow cytometry, immunohistochemistry)

Studies have shown that effective knockdown of PSMC5 leads to measurable phenotypes, including reduced cancer cell proliferation and invasion , altered immune cell infiltration , and attenuation of LPS-induced neuroinflammation .

How does PSMC5 interact with TLR4 signaling pathways in neuroinflammation?

PSMC5 plays a crucial role in regulating neuroinflammation through its interaction with Toll-like receptor 4 (TLR4) signaling:

• Direct protein interaction: PSMC5 physically interacts with TLR4 via specific amino acid residues (Glu284, Met139, Leu127, and Phe283) . This interaction appears to be a key regulatory mechanism in TLR4-mediated inflammatory responses.

• Effects on microglial polarization:

  • PSMC5 knockdown shifts microglial polarization from pro-inflammatory to anti-inflammatory phenotypes

  • This effect is abolished in TLR4-knockout mice and with TLR4 inhibitor treatment, suggesting PSMC5 functions through TLR4

• Downstream signaling regulation:

  • PSMC5 knockdown attenuates LPS-induced phosphorylation of IκB-α and p65 (key components of NF-κB signaling)

  • This leads to reduced expression of pro-inflammatory factors (TNF-α, IL-1β, PGE2, NO, iNOS, COX-2)

  • Simultaneously, PSMC5 knockdown upregulates anti-inflammatory mediators (IL-4, IL-10)

• Functional outcomes:

  • In mouse models, PSMC5 knockdown attenuates LPS-induced cognitive and motor impairments

  • PSMC5 knockdown restores synaptic ultrastructure and protein levels disrupted by LPS

These findings suggest that targeting PSMC5-TLR4 interaction could be a potential therapeutic strategy for neuroinflammatory conditions. Researchers studying PSMC5 in neuroinflammation should consider examining both TLR4-dependent and independent pathways to fully understand its regulatory mechanisms.

How can researchers distinguish between PSMC5's proteasomal and non-proteasomal functions?

PSMC5 has both proteasome-dependent and independent functions, which can be distinguished through several experimental approaches:

• Selective inhibition strategies:

  • Proteasome inhibitors (MG132, bortezomib): These affect all proteasome functions but can help determine if observed phenotypes are proteasome-dependent

  • PSMC5-specific point mutations: Creating mutations that affect PSMC5's ATPase activity versus its protein-interaction domains can separate its functions

• Protein-protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry to identify PSMC5 binding partners outside the proteasome complex

  • Proximity labeling techniques (BioID, APEX) to identify proteins in PSMC5's vicinity under different conditions

• Subcellular localization:

  • Immunofluorescence microscopy to determine if PSMC5 localizes to non-proteasomal sites

  • Cell fractionation followed by Western blotting to quantify PSMC5 distribution across cellular compartments

• Comparative studies:

  • Compare phenotypes from PSMC5 knockdown with knockdown of other proteasome subunits

  • Effects unique to PSMC5 depletion likely represent non-proteasomal functions

• Rescue experiments:

  • Express PSMC5 mutants lacking specific domains in PSMC5-knockdown cells

  • Determine which domains are required for specific functions

This systematic approach can help researchers attribute observed phenotypes to either PSMC5's role in the proteasome or its independent functions in processes like transcriptional regulation and immune signaling.

What methodologies are most appropriate for studying PSMC5's role in immune cell infiltration?

To effectively study PSMC5's role in regulating immune cell infiltration, researchers should consider a multi-faceted approach:

• In silico analyses:

  • Single-cell RNA sequencing data analysis to correlate PSMC5 expression with immune cell populations

  • GSVA (Gene Set Variation Analysis) and GSEA (Gene Set Enrichment Analysis) to identify enriched immune-related pathways

  • ssGSEA (single-sample Gene Set Enrichment Analysis) to evaluate correlations between PSMC5 and immune infiltrating cells in the tumor microenvironment

• In vitro co-culture systems:

  • Co-culture of PSMC5-manipulated cancer cells with immune cells (T cells, B cells, macrophages, neutrophils)

  • Transwell migration assays to assess immune cell recruitment

  • Flow cytometry to analyze immune cell phenotypes (M1/M2 macrophages, N1/N2 neutrophils)

• In vivo models:

  • Conditional knockout of PSMC5 in specific immune cell populations

  • Analysis of immune infiltration in tumor models with PSMC5 manipulation

  • Multiplex immunohistochemistry to visualize multiple immune cell types simultaneously

• Chemokine profiling:

  • Measure expression of chemokines associated with PSMC5 (CCL3, CCL4, CCL5)

  • Neutralizing antibody experiments to block specific chemokines

• Mechanistic validation:

  • ChIP-seq to identify PSMC5's direct transcriptional targets related to immune function

  • RNA-seq of sorted immune cells after PSMC5 manipulation

  • Pathway inhibition studies to determine if PSMC5's effects on immune infiltration depend on specific signaling pathways

This comprehensive methodology allows researchers to not only identify correlations between PSMC5 and immune infiltration but also elucidate the underlying mechanisms and functional consequences.

What are the optimal conditions for PSMC5 antibody use in various applications?

Optimizing PSMC5 antibody usage varies by application:

• Western Blotting:

  • Recommended dilution: 1:500 - 1:2000

  • Sample preparation: Complete lysis buffers containing protease inhibitors

  • Blocking: 5% non-fat milk or BSA in TBST

  • Detection: HRP-conjugated secondary antibodies with ECL detection systems

• Immunohistochemistry (IHC-P):

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Blocking: 10% normal serum from secondary antibody host species

  • Primary antibody incubation: Overnight at 4°C

  • Detection: Polymer-based detection systems for enhanced sensitivity

• Immunocytochemistry/Immunofluorescence (ICC/IF):

  • Fixation: 4% paraformaldehyde (10-15 minutes)

  • Permeabilization: 0.1-0.3% Triton X-100 in PBS

  • Blocking: 1-5% BSA in PBS

  • Primary antibody incubation: 1-3 hours at room temperature or overnight at 4°C

  • Counterstaining: DAPI for nuclear visualization

• ELISA:

  • Coating: Optimize antibody concentration for coating (typically 1-10 μg/ml)

  • Sample dilution: Establish standard curves with recombinant PSMC5

  • Detection: HRP or AP-conjugated detection systems

Each application requires optimization with positive control samples (HepG2, MCF7, HeLa, 293T, NIH/3T3) to determine the ideal conditions for specific experimental setups.

How can researchers troubleshoot common issues with PSMC5 antibody experiments?

When troubleshooting PSMC5 antibody experiments, researchers should systematically address these common issues:

• Weak or no signal in Western blotting:

  • Increase antibody concentration or incubation time

  • Verify protein transfer by Ponceau S staining

  • Use fresh sample preparation with protease inhibitors

  • Ensure PSMC5 is expressed in your sample by checking positive controls like HepG2 or HeLa cells

• High background in immunohistochemistry/immunofluorescence:

  • Increase blocking time or concentration

  • Reduce primary and secondary antibody concentrations

  • Include additional washing steps

  • Use more specific secondary antibodies

• Non-specific bands in Western blotting:

  • Increase blocking stringency

  • Optimize antibody dilution

  • Use gradient gels for better separation

  • Consider alternative antibodies targeting different epitopes of PSMC5

• Inconsistent results across experiments:

  • Standardize protein extraction methods

  • Use the same positive controls across experiments

  • Document lot numbers of antibodies used

  • Maintain consistent incubation times and temperatures

• False positives/negatives:

  • Validate with genetic approaches (PSMC5 knockdown/overexpression)

  • Use multiple antibodies targeting different epitopes

  • Include appropriate controls (isotype, no primary)

  • Consult literature for known issues with specific antibodies

Methodical troubleshooting with appropriate controls helps ensure reliable and reproducible results when working with PSMC5 antibodies.

How can researchers effectively study PSMC5's role in cancer biomarker development?

To investigate PSMC5 as a potential cancer biomarker, researchers should implement these methodological approaches:

• Multi-cohort validation studies:

  • Analyze PSMC5 expression across multiple independent patient cohorts

  • Use both public datasets (TCGA, GEO) and institution-specific cohorts

  • Perform detailed clinical correlation analyses with survival, stage, and treatment response

• Multi-omics integration:

  • Correlate PSMC5 protein expression with mRNA levels

  • Integrate with mutation data, methylation patterns, and copy number alterations

  • Perform pathway enrichment analyses to understand biological context

• Biomarker performance metrics:

  • Calculate sensitivity, specificity, positive/negative predictive values

  • Generate ROC curves to assess diagnostic potential

  • Perform multivariate analyses to determine independent prognostic value

• Comparison with established biomarkers:

  • Compare PSMC5's performance against standard clinical biomarkers

  • Evaluate additive value in multi-marker panels

  • Assess correlation with immune checkpoint markers like PD-L1, CTLA-4, and IDO1

• Predictive biomarker assessment:

  • Evaluate PSMC5 expression in pre- and post-treatment samples

  • Correlate with treatment response, particularly to immunotherapies

  • Develop predictive models incorporating PSMC5 expression

• Technical validation:

  • Standardize antibody-based detection methods

  • Develop quantitative assays (ELISA, digital PCR)

  • Evaluate detectability in liquid biopsies (circulating tumor cells, cell-free DNA)

As demonstrated in colorectal cancer research, PSMC5 expression correlates with poorer prognosis and may serve as a predictive biomarker for immunotherapy response , making these methodological approaches particularly relevant.

What experimental designs are most appropriate for investigating PSMC5's role in microglial polarization?

To thoroughly investigate PSMC5's role in microglial polarization, researchers should consider these experimental designs:

• In vitro models:

  • Primary microglial cultures and microglial cell lines (BV2)

  • PSMC5 manipulation through siRNA, shRNA, or CRISPR techniques

  • Stimulation with polarizing factors (LPS for M1, IL-4/IL-13 for M2)

  • Analysis of inflammatory mediators (TNF-α, IL-1β, IL-4, IL-10)

• Ex vivo approaches:

  • Acute brain slices from wild-type and PSMC5-modified animals

  • Organotypic slice cultures with microglial manipulation

  • Two-photon imaging to visualize microglial dynamics

• In vivo models:

  • Conditional PSMC5 knockout in microglia (Cx3cr1-Cre or Tmem119-Cre)

  • LPS-induced neuroinflammation models

  • Disease-specific models (Alzheimer's, Parkinson's, stroke)

  • Behavioral testing to correlate microglial phenotypes with cognitive/motor functions

• Mechanistic investigation:

  • TLR4 pathway analysis (IκB-α and p65 phosphorylation)

  • Co-immunoprecipitation to confirm PSMC5-TLR4 interaction

  • Site-directed mutagenesis of interaction sites (Glu284, Met139, Leu127, Phe283)

  • Pharmacological intervention (TLR4 inhibitors, proteasome inhibitors)

• Comprehensive phenotyping:

  • Flow cytometry for surface marker profiling

  • Single-cell RNA sequencing to identify microglial subtypes

  • Spatial transcriptomics to understand regional differences

  • Multiplex cytokine assays for secretome analysis

• Translational relevance:

  • Analysis of human microglial samples from patients with neuroinflammatory conditions

  • Correlation of findings with clinical parameters

  • Testing PSMC5-targeting compounds in preclinical models

This multi-faceted approach enables researchers to comprehensively understand how PSMC5 regulates microglial polarization and identify potential therapeutic interventions for neuroinflammatory conditions.

How might PSMC5 function as a therapeutic target in different disease contexts?

PSMC5's diverse functions suggest several therapeutic targeting strategies across disease contexts:

• Cancer therapy approaches:

  • Small molecule inhibitors targeting PSMC5's ATPase activity

  • Peptide-based disruptors of PSMC5-protein interactions

  • Combination with immune checkpoint inhibitors, as PSMC5 correlates with immune checkpoint molecules

  • Targeted protein degradation approaches (PROTACs) specific to PSMC5

• Neuroinflammatory disease applications:

  • Compounds disrupting PSMC5-TLR4 interaction

  • Delivery systems targeting microglia for PSMC5 modulation

  • Repurposing existing drugs that may affect PSMC5 function

• Target validation considerations:

  • Inducible and cell-type specific genetic models

  • Humanized mouse models for translational relevance

  • Patient-derived organoids for personalized testing

• Biomarker-guided therapy:

  • PSMC5 expression as a stratification marker for clinical trials

  • Monitoring PSMC5 activity as a pharmacodynamic marker

  • Combined targeting of PSMC5 with pathway-specific inhibitors

• Delivery challenges and solutions:

  • Nanoparticle-based delivery of PSMC5 modulators

  • Blood-brain barrier penetration for neurological applications

  • Tissue-specific delivery systems to minimize systemic effects

• Potential therapeutic resistance mechanisms:

  • Compensatory upregulation of other proteasome subunits

  • Alternative pathway activation

  • Strategies to overcome resistance through combination approaches

The therapeutic potential of targeting PSMC5 stems from its involvement in multiple cellular processes and disease mechanisms, from cancer progression to neuroinflammation , offering diverse opportunities for intervention development.

What are the most promising techniques for studying PSMC5 protein-protein interactions?

To comprehensively study PSMC5's protein-protein interactions, researchers should consider these cutting-edge techniques:

• Affinity-based approaches:

  • Co-immunoprecipitation with PSMC5-specific antibodies

  • Tandem affinity purification with tagged PSMC5

  • Proximity-dependent biotinylation (BioID, TurboID)

  • APEX2-based proximity labeling

• Structural biology methods:

  • Cryo-electron microscopy of PSMC5-containing complexes

  • X-ray crystallography of PSMC5 with interacting domains

  • NMR spectroscopy for dynamic interaction studies

  • Hydrogen-deuterium exchange mass spectrometry

• Live-cell interaction analysis:

  • Fluorescence resonance energy transfer (FRET)

  • Bioluminescence resonance energy transfer (BRET)

  • Split-fluorescent/luciferase complementation assays

  • Optogenetic approaches for inducible interactions

• High-throughput screening:

  • Yeast two-hybrid or mammalian two-hybrid screens

  • Protein microarrays with purified PSMC5

  • CRISPR screens for genetic modifiers of PSMC5 function

• Computational approaches:

  • Molecular docking simulations

  • Molecular dynamics simulations of PSMC5-protein complexes

  • Machine learning predictions of interaction partners

• Validation and characterization:

  • Site-directed mutagenesis of interaction interfaces

  • Peptide competition assays

  • Domain mapping through truncation constructs

  • Functional consequences of disrupting specific interactions

Application of these techniques has already revealed important PSMC5 interactions, such as its binding to TLR4 through specific amino acid residues (Glu284, Met139, Leu127, and Phe283) . Future studies may uncover additional interaction partners involved in cancer progression and other cellular processes, potentially identifying novel therapeutic targets.