PSMC5 Antibody, FITC conjugated

What is PSMC5 and why is it significant for research?

PSMC5 (proteasome 26S subunit, ATPase 5) is a 19S regulatory component of the proteasome that recognizes ubiquitin-labeled proteins and processes them for degradation by the 20S complex. Beyond its proteolytic functions, PSMC5 has emerging roles in transcriptional regulation through non-proteolytic mechanisms . Research significance stems from its involvement in critical cellular processes including inflammation, cancer progression, and signal transduction. PSMC5 interacts directly with TLR4 to regulate neuroinflammation , promotes proliferation and metastasis in colorectal cancer , and modulates ERK1/2 signaling through interaction with the scaffold protein Shoc2 .

How do FITC-conjugated PSMC5 antibodies work in cellular imaging applications?

FITC-conjugated PSMC5 antibodies function through direct fluorescence detection, eliminating the need for secondary antibodies in imaging workflows. The antibody's specificity for PSMC5 epitopes combined with FITC's excitation maximum at approximately 495 nm and emission maximum around 519 nm allows for visualization using standard fluorescence microscopy filters. For optimal results in cellular imaging, researchers should perform fixation and permeabilization steps appropriate to the cellular compartment where PSMC5 is being studied. The antibody can effectively visualize PSMC5's subcellular distribution, which varies depending on cellular context - from cytoplasmic proteasome complexes to endosomal localization when interacting with scaffold proteins like Shoc2 .

What fixation methods are optimal for PSMC5 immunofluorescence studies?

Optimal fixation for PSMC5 immunofluorescence depends on the specific cellular compartment being investigated. For general PSMC5 detection, 4% paraformaldehyde fixation for 10-15 minutes at room temperature preserves both protein structure and cellular architecture. When studying PSMC5 interactions with membrane proteins like TLR4, a gentler fixation with 2% paraformaldehyde may better preserve membrane structures and protein complexes. If investigating PSMC5's associations with the cytoskeleton or endosomal compartments, a brief (5 minute) methanol fixation at -20°C may enhance epitope accessibility. Critical controls should include validating specificity through siRNA knockdown of PSMC5, as demonstrated in studies examining PSMC5-Shoc2 endosomal localization .

How can FITC-conjugated PSMC5 antibodies be used to study endosomal trafficking of PSMC5-containing complexes?

To study PSMC5's role in endosomal trafficking, researchers can employ dual-labeling approaches using FITC-conjugated PSMC5 antibodies alongside markers for different endosomal compartments. This approach has revealed that PSMC5 triggers translocation of the Shoc2 scaffold complex to late endosomes and/or multivesicular bodies (MVBs) . For optimal experimental design:

• Co-stain with markers such as Rab5 (early endosomes), Rab7 (late endosomes), or CD63 (MVBs) using antibodies with non-overlapping fluorophores

• Apply fractionation techniques to isolate crude endosomal fractions, as demonstrated in studies where PSMC5-Shoc2 complexes were identified at endosomal interfaces

• Implement live-cell imaging with FITC-PSMC5 antibody fragments to track dynamic changes in PSMC5 localization upon stimulation

• Validate endosomal localization using subcellular fractionation followed by western blotting, comparing crude endosomal fractions with Golgi and ER interfaces

This methodology has revealed that PSMC5 oligomerization is necessary for targeting Shoc2 to endosomes, a critical aspect of ERK1/2 signal modulation .

What controls and validation steps are essential when using PSMC5 antibodies in neuroinflammation research?

When investigating PSMC5's role in neuroinflammation, several critical validation steps ensure experimental rigor:

• Antibody specificity: Validate through western blotting of samples with PSMC5 knockdown (siRNA/shRNA) compared to controls

• Positive controls: Include LPS-stimulated microglial cells, which show increased PSMC5 expression in a time-dependent manner

• Negative controls: Use samples treated with PSMC5 inhibitors or from knockdown models

• Concentration gradient testing: Determine optimal antibody concentration to avoid non-specific binding

• Cross-validation: Compare FITC-conjugated antibody results with unconjugated primary antibodies detected by secondary methods

• Functional validation: Confirm that detected changes in PSMC5 localization correlate with alterations in inflammatory markers (IL-1β, COX-2, PGE2)

These controls are particularly important when studying PSMC5's interaction with TLR4, which mediates microglial activation in neuroinflammatory conditions, as PSMC5 has been shown to bind directly to TLR4 through specific residues (Glu284, Met139, Leu127, and Phe283) .

How can multiplexed flow cytometry with FITC-PSMC5 antibodies be optimized for tumor-infiltrating immune cell analysis?

For multiplexed flow cytometry analyzing PSMC5 expression in tumor microenvironments:

• Panel design: Combine FITC-PSMC5 antibody with markers for:

  • Tumor cells (e.g., EpCAM)

  • Immune cell populations (CD8+ T cells, B cells, macrophages, neutrophils)

  • Functional markers (activation, exhaustion)

• Compensation: Carefully compensate for FITC spillover into other channels, particularly PE

• PSMC5 expression analysis stratification:

  • Compare PSMC5 levels across different immune populations

  • Correlate with chemokine expression (CCL3, CCL4, CCL5) which are positively associated with PSMC5 expression

• Cell sorting validation:

  • Sort PSMC5-high versus PSMC5-low populations

  • Perform functional assays to confirm biological differences

This approach can reveal how PSMC5 expression correlates with infiltration of specific immune cell types, as research has shown PSMC5 negatively correlates with CD8+ T cells and B cells while promoting macrophage and neutrophil infiltration in colorectal cancer .

What factors influence the detection sensitivity of PSMC5 in different subcellular compartments?

Detection sensitivity of PSMC5 varies across subcellular compartments due to several factors:

• Epitope accessibility: PSMC5's conformation changes when engaged in different protein complexes, potentially masking antibody binding sites. When studying PSMC5 in proteasome complexes versus its non-proteolytic functions, different antibody clones targeting distinct epitopes may be required.

• Fixation impact: Overfixation can reduce signal by crosslinking epitopes, while underfixation risks protein loss. For endosomal PSMC5 detection, mild fixation (2% paraformaldehyde for 10 minutes) followed by gentle permeabilization (0.1% saponin) preserves structure while maintaining antibody accessibility.

• Protein complex dissociation: PSMC5 interaction with partners like Shoc2 and TLR4 may require specialized buffers during sample preparation. When analyzing PSMC5-TLR4 interactions, use of DSP (dithiobis(succinimidyl propionate)) crosslinking prior to lysis helps preserve complex integrity .

• Signal amplification: For detecting low PSMC5 levels, particularly on endosomes where standard methods show limitations, tyramide signal amplification can increase sensitivity while maintaining specificity .

How should researchers address inconsistencies in PSMC5 detection between immunofluorescence and biochemical assays?

When facing discrepancies between imaging and biochemical data:

• Epitope masking assessment: Different experimental conditions may affect epitope exposure differently. Perform epitope retrieval optimization for immunofluorescence using methods like heat-induced epitope retrieval or different detergents.

• Antibody validation across methods: Verify that the FITC-conjugated antibody performs consistently in both applications by testing multiple antibody clones against different PSMC5 epitopes.

• Sample preparation harmonization: Align lysis buffers for biochemical assays with fixatives for immunofluorescence. For instance, when studying PSMC5 in endosomal fractions, comparable extraction conditions should be used for both western blotting and microscopy .

• Quantification approach correlation: Develop calibration curves that correlate fluorescence intensity in imaging with protein quantity in western blots using purified PSMC5 standards.

• Context-dependent expression: Consider that PSMC5 expression and detection may vary with cell cycle stage, activation state, or stress conditions. Synchronize cells when possible and document experimental conditions thoroughly.

How can FITC-conjugated PSMC5 antibodies be used to investigate PSMC5's role in ERK1/2 signaling regulation?

To investigate PSMC5's function in ERK1/2 pathway regulation:

• Live-cell imaging approach:

  • Transfect cells with fluorescently-tagged signaling partners (Shoc2-tRFP)

  • Apply FITC-PSMC5 antibody fragments for dynamic tracking

  • Monitor real-time changes in co-localization following pathway stimulation

• Co-immunoprecipitation workflow:

  • Use FITC-conjugated PSMC5 antibodies for direct immunoprecipitation

  • Analyze co-precipitated proteins including Shoc2, RAF-1, and HUWE1

  • Compare complex composition between unstimulated and stimulated conditions

• Subcellular fractionation analysis:

  • Isolate crude endosomal fractions versus cytosolic components

  • Quantify PSMC5 redistribution during signaling events

  • Correlate with ERK1/2 phosphorylation levels

This approach can reveal how PSMC5 mediates displacement of the E3 ligase HUWE1 from the Shoc2 scaffolding complex, attenuating ubiquitylation of Shoc2 and RAF-1, with corresponding changes in ERK1/2 activity .

What are the appropriate experimental designs to study the relationship between PSMC5 and TLR4 in neuroinflammation?

For investigating PSMC5-TLR4 interactions in neuroinflammation:

• Molecular interaction confirmation:

  • Conduct co-immunoprecipitation with FITC-PSMC5 antibodies followed by TLR4 western blotting

  • Perform proximity ligation assays to visualize endogenous protein interactions

  • Validate specific binding residues (Glu284, Met139, Leu127, Phe283) through site-directed mutagenesis

• Functional analysis workflow:

  • Transfect cells with wild-type or mutant PSMC5 constructs

  • Stimulate with LPS to activate TLR4 signaling

  • Measure inflammatory markers (iNOS, COX-2, NO, PGE2)

  • Correlate inflammatory response with PSMC5-TLR4 binding capability

• In vivo models approach:

  • Deliver shRNA PSMC5 intracerebroventricularly before LPS administration

  • Conduct behavioral tests and biochemical assays to evaluate neuroprotection

  • Compare with TLR4 inhibitor treatments (VIPER) to establish pathway specificity

This comprehensive approach has demonstrated that PSMC5 regulates neuroinflammation by directly binding to TLR4, affecting TLR4-mediated MyD88-dependent signaling pathways in vivo and in vitro .

How can researchers quantitatively assess PSMC5's impact on tumor-infiltrating immune cells using FITC-conjugated antibodies?

For quantitative assessment of PSMC5's impact on tumor immunity:

• Multi-parameter flow cytometry methodology:

  • Process tumor samples into single-cell suspensions

  • Stain with FITC-PSMC5 alongside markers for:

    • T cells (CD3, CD8, CD4, PD-1)

    • B cells (CD19, CD20)

    • Macrophages (CD68, CD163)

    • Neutrophils (CD66b)

  • Gate on PSMC5-high versus PSMC5-low tumor cells

  • Quantify immune cell frequencies and activation states in each population

• Spatial analysis protocol:

  • Perform multiplexed immunofluorescence on tissue sections

  • Measure distances between PSMC5+ cells and various immune populations

  • Calculate spatial correlation indices

• Functional correlation:

  • Sort PSMC5-high versus PSMC5-low tumor cells

  • Perform chemokine expression analysis (CCL3, CCL4, CCL5)

  • Conduct conditioned media experiments to assess immune cell migration

This approach enables correlation between PSMC5 expression levels and specific immune cell populations, consistent with findings that PSMC5 negatively correlates with CD8+ T cells and B cells while promoting macrophage and neutrophil infiltration in colorectal cancer .

How can researchers combine PSMC5 antibody labeling with proteomics to identify novel interaction partners?

To identify novel PSMC5 interaction partners:

• Immunoprecipitation-mass spectrometry workflow:

  • Use FITC-PSMC5 antibodies for immunoprecipitation

  • Perform on-bead digestion to minimize contamination

  • Analyze by LC-MS/MS with appropriate controls

  • Filter candidates using statistical significance thresholds

• Proximity-dependent biotinylation approach:

  • Create PSMC5-BioID or PSMC5-TurboID fusion proteins

  • Express in relevant cell systems under physiological conditions

  • Capture biotinylated proteins in proximity to PSMC5

  • Validate hits with reciprocal FITC-PSMC5 co-localization

• Dynamic interaction analysis:

  • Compare interactomes under different conditions (e.g., LPS stimulation, tumor microenvironment)

  • Map interaction networks using computational tools

  • Validate key interactions using FITC-PSMC5 co-localization

This approach follows the precedent set in studies that identified PSMC5's interaction with Shoc2 through yeast two-hybrid screening and confirmed it through multiple validation methods including GST pull-down assays and co-immunoprecipitation .

What methodological approaches can address data contradictions in PSMC5 subcellular localization studies?

When facing contradictory data regarding PSMC5 localization:

• Resolution enhancement protocol:

  • Implement super-resolution microscopy (STED, STORM, or SIM)

  • Compare with conventional confocal microscopy results

  • Quantify co-localization coefficients using standardized algorithms

• Temporal dynamics assessment:

  • Conduct time-course experiments following stimulation

  • Track PSMC5 movement through different compartments

  • Correlate temporal patterns with functional outcomes

• Context-dependent localization analysis:

  • Compare PSMC5 distribution across cell types (microglia vs. cancer cells)

  • Assess impact of differentiation states or activation conditions

  • Evaluate influence of interacting partners (Shoc2, TLR4) on localization

• Fractionation validation:

  • Perform detailed subcellular fractionation with gradient centrifugation

  • Analyze PSMC5 distribution across all fractions

  • Compare biochemical data with imaging findings