Cysteine protease inhibitor 1 Antibody

What exactly is Cysteine protease inhibitor 1 and why is it significant in research?

Cysteine protease inhibitor 1 belongs to the cystatin superfamily, functioning primarily by binding to cysteine proteases (such as cathepsins B/L) to inhibit their enzymatic activity. Its significance stems from its involvement in critical biological processes including cancer progression, immune regulation, and parasitic infections. In esophageal squamous cell carcinoma (ESCC), CST1 (a human CPI-1) has been identified as having oncogenic properties by enhancing mitochondrial respiratory chain complex I activity to activate the OXPHOS/MEK/ERK axis, consequently promoting cancer metastasis . In parasites like Baylisascaris schroederi, BsCPI-1 aids survival by modulating host immune responses through TLR2/4 signaling pathways . Understanding CPI-1's structure-function relationships provides insights into proteolytic regulation across normal physiology and pathological conditions.

How do I distinguish between different types of cysteine protease inhibitors in my experimental design?

Distinguishing between different cysteine protease inhibitors requires a multi-faceted approach:

• Structural analysis: CPI-1 typically contains conserved domains characteristic of the cystatin superfamily. Analyze primary sequence for QxVxG motifs and confirm with structural predictions.

• Inhibitory profile assessment: Evaluate inhibitory activity against specific proteases using fluorogenic substrates. For example, when characterizing rBsCPI-1, researchers used Z-Phe-Arg-AMC for papain and cathepsin L, and Z-Arg-Arg-AMC for cathepsin B .

• Dose-response analysis: Determine IC₅₀ values by incubating varying concentrations (0-100 nM) of your inhibitor with target proteases (20 nM) and measure residual proteolytic activity .

• Reversibility testing: Distinguish between reversible inhibitors (like most cystatins) and irreversible inhibitors by monitoring enzyme activity recovery after dilution.

• Comparative analysis: Create inhibitory profiles against a panel of proteases, as CPI-1 typically shows preferential inhibition of cathepsin L over cathepsin B .

This systematic characterization will provide a definitive inhibitory signature that differentiates your CPI from other cysteine protease inhibitors.

What are the current applications of CPI-1 antibodies in cancer research?

CPI-1 antibodies have emerged as important tools in cancer research, particularly for understanding metastatic mechanisms:

• Biomarker identification: CST1 has been identified as aberrantly expressed in multiple cancers, including breast, lung, liver, gastric, and esophageal cancers . CPI-1 antibodies enable detection of this potential biomarker in tissue samples and serum.

• Metastasis pathway elucidation: Antibodies against CST1 have revealed its role in enhancing mitochondrial complex I enzyme activity through interaction with GRIM19 protein, consequently elevating oxidative phosphorylation (OXPHOS) levels in ESCC . This mechanism activates the MEK/ERK pathway, promoting cancer cell migration and invasion.

• Therapeutic target validation: Immunoprecipitation studies using CPI-1 antibodies have demonstrated protein-protein interactions between CST1 and components of the respiratory chain complex I, validating CST1/OXPHOS as a promising therapeutic target .

• Regulatory network mapping: Western blot analysis using CST1 antibodies has revealed a reciprocal regulatory relationship between OXPHOS and the MEK/ERK pathway in ESCC cells, with inhibition of either pathway affecting the other .

When designing experiments, researchers should incorporate appropriate controls (isotype controls, blocking peptides) and validate antibody specificity through knockdown/knockout approaches to ensure reliable interpretation of results.

How are CPI-1 antibodies utilized in parasitology and infectious disease research?

CPI-1 antibodies serve as critical tools in parasitology research through multiple applications:

• Identification of parasite secretory products: CPI-1 antibodies enable detection of cysteine protease inhibitors in excretory/secretory (ES) antigens from parasites. For example, anti-rBsCPI-1 sera specifically recognize a 34.53 kDa band in B. schroederi ES antigens, confirming its secretion into host environments .

• Immunomodulation mechanism studies: CPI-1 antibodies help elucidate how parasitic CPIs manipulate host immune responses. Research has demonstrated that rBsCPI-1 induces macrophage polarization to the M2 subtype via TLR2/4 signaling, suppressing pro-inflammatory markers (IL-12β, iNOS) while upregulating anti-inflammatory markers (CD206, IL-10) .

• Host-parasite interaction analysis: Through immunofluorescence studies, researchers have used CPI-1 antibodies to track the trafficking and localization of parasite-derived CPIs in host cells, revealing their impact on antigen presentation machinery .

• Therapeutic target identification: Immunodetection of CPI-1 expression across different parasite life stages helps identify optimal timing for therapeutic intervention. Studies have shown that cysteine protease inhibitors can selectively kill parasites at therapeutic doses without significant host toxicity .

When designing these experiments, include negative control sera (pre-immunization) and confirm antibody specificity through Western blot analysis against recombinant protein to ensure accurate interpretation of results.

What are the optimal conditions for using CPI-1 antibodies in Western blot applications?

Optimizing Western blot protocols for CPI-1 antibodies requires attention to several critical parameters:

• Sample preparation:

  • For tissue/cell lysates: Use RIPA buffer with 1% PMSF to effectively extract CPI-1 proteins

  • For parasite ES products: Concentrate using PEG2000 before filtration through 0.22μm filters

• SDS-PAGE conditions:

  • Use 12% polyacrylamide gels for optimal resolution of CPI-1 (~12-35 kDa depending on source)

  • Load 50μg protein per lane for cell/tissue lysates

• Transfer parameters:

  • Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

  • Use nitrocellulose membranes for better protein binding and lower background

• Blocking conditions:

  • 5% skim milk in TBST for 2 hours at room temperature to minimize non-specific binding

• Antibody dilutions and incubation:

  • Primary antibody: 1:1000-1:2000 dilution in 5% skim milk/TBST overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-species IgG at 1:5000 for 1 hour at room temperature

• Detection system:

  • Use ultrasensitive ECL chemiluminescence reagent for optimal signal-to-noise ratio

• Controls:

  • Positive control: Recombinant CPI-1 protein (commercially available or lab-produced)

  • Negative control: Pre-immune serum at same dilution as primary antibody

When troubleshooting, adjust antibody concentrations and incubation times first, followed by modifications to blocking conditions if background issues persist.

How can I optimize co-immunoprecipitation experiments to study CPI-1 protein interactions?

Co-immunoprecipitation (Co-IP) is crucial for elucidating CPI-1 protein-protein interactions, particularly with targets like GRIM19 in mitochondrial pathways . To optimize this technique:

• Lysate preparation:

  • Use gentle lysis buffers (e.g., 50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40) supplemented with protease inhibitors

  • For mitochondrial proteins, include specific extraction steps: initial homogenization followed by differential centrifugation to isolate mitochondrial fractions

  • Verify protein concentration using BCA assay; aim for 1-2 mg/ml total protein

• Pre-clearing step:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation (2500×g, 5 min) to reduce non-specific binding

• Antibody-bead coupling:

  • Incubate 2-5μg anti-CPI-1 antibody with Protein A/G beads for 1 hour

  • Wash beads 3× with lysis buffer to remove unbound antibody

• Immunoprecipitation:

  • Incubate pre-cleared lysate with antibody-coupled beads overnight at 4°C with gentle rotation

  • Include parallel samples with isotype control antibodies or pre-immune serum

  • For interaction with GRIM19, increase incubation time to 16-18 hours

• Washing and elution:

  • Perform 4-5 sequential washes with decreasing salt concentration buffers

  • Elute bound proteins with 0.1M glycine (pH 2.5) or by boiling in SDS sample buffer

• Analysis:

  • Analyze by SDS-PAGE followed by western blotting for both CPI-1 and suspected interaction partners

  • Validate results with reciprocal Co-IP (using antibodies against the interaction partner)

This approach has successfully demonstrated interaction between CST1 and GRIM19, a critical component of mitochondrial complex I .

What is the recommended protocol for measuring inhibitory activity of CPI-1 against target proteases?

To accurately measure the inhibitory activity of CPI-1 against cysteine proteases, implement this optimized protocol based on established research methodologies :

• Buffer preparation:

  • For papain: 100mM sodium acetate (pH 6.0), 1mM EDTA, 2mM dithiothreitol

  • For cathepsins B/L: 100mM sodium acetate (pH 5.0), 1mM EDTA, 1mM DTT

• Protease preparation:

  • Dilute purified proteases (papain, cathepsin B, cathepsin L) to 20nM final concentration

  • Reconstitute lyophilized enzymes in appropriate buffer with brief activation period (10 minutes at room temperature)

• Inhibitor pre-incubation:

  • Prepare CPI-1 dilution series (0-100nM) in reaction buffer

  • Pre-incubate proteases with CPI-1 for 30 minutes at 37°C before substrate addition

• Substrate preparation:

  • For papain and cathepsin L: Z-Phe-Arg-AMC (10μM final concentration)

  • For cathepsin B: Z-Arg-Arg-AMC (10μM final concentration)

• Reaction monitoring:

  • After adding substrate, incubate reaction mixture at 37°C for 1 hour

  • Monitor fluorescence at excitation/emission wavelengths of 380/460nm

  • Take measurements at 5-minute intervals for kinetic analysis

• Controls and standards:

  • Positive control: E64 (10nM final concentration)

  • Negative control: BSA at equivalent concentration to CPI-1

  • Enzyme-only control: Protease without inhibitor

• Data analysis:

  • Calculate percent inhibition relative to enzyme-only control

  • Determine IC₅₀ values by plotting inhibition percentage against inhibitor concentration

  • For K_i calculation, use Lineweaver-Burk or Dixon plots with varying substrate concentrations

This methodology enables precise characterization of inhibitory profiles and comparison between different CPI variants or mutants.

How do I interpret conflicting results about CPI-1 function across different experimental systems?

Interpreting conflicting CPI-1 function data requires systematic analysis of experimental variables and biological context:

• Source variation analysis:

  • Compare sequence homology between CPI-1 from different species (human CST1 vs. parasite BsCPI-1)

  • Examine post-translational modifications that might affect function

  • Consider recombinant vs. native protein differences, particularly regarding glycosylation patterns

• Experimental design differences:

  • Compare inhibitor concentrations used across studies (therapeutic effects observed at 5-50μM range)

  • Analyze buffer conditions, especially pH variations that affect cysteine protease activity

  • Evaluate cell type-specific responses (e.g., cancer cells vs. immune cells)

• Context-dependent functions:

  • In cancer contexts: CPI-1 promotes metastasis via OXPHOS/MEK/ERK pathway activation

  • In parasitic infections: CPI-1 modulates immune responses via TLR2/4 signaling

  • These opposing functions reflect biological adaptations in different systems

• Methodological resolution:

  • For contradictory enzymatic inhibition results: Standardize enzyme:inhibitor ratios

  • For conflicting cellular responses: Perform time-course experiments to capture temporal dynamics

  • For opposing in vivo outcomes: Consider route of administration differences and pharmacokinetics

• Validation strategy:

  • Implement genetic approaches (siRNA, CRISPR) alongside inhibitor studies

  • Use multiple antibody clones targeting different epitopes

  • Perform rescue experiments with wild-type protein after knockdown

This systematic approach will help reconcile apparently contradictory findings and develop a more nuanced understanding of CPI-1's context-dependent functions.

What are the common technical challenges when working with CPI-1 antibodies and how can they be addressed?

Researchers face several technical challenges when working with CPI-1 antibodies that can be addressed through specific optimization strategies:

• Cross-reactivity issues:

  • Challenge: CPI-1 belongs to the cystatin superfamily with conserved structural motifs, potentially causing cross-reactivity

  • Solution: Perform pre-absorption with related cystatins; validate specificity using knockout/knockdown samples; employ epitope mapping to select antibodies targeting unique regions

• Sensitivity limitations:

  • Challenge: Low endogenous expression levels in certain cell types or tissues

  • Solution: Implement signal amplification methods (TSA for IHC); use ultrasensitive ECL detection systems; concentrate samples before immunoblotting

• Conformational epitope recognition:

  • Challenge: Antibodies may not recognize native protein conformation in certain applications

  • Solution: Use different fixation protocols for IHC/ICC; test multiple antibody clones; consider native versus denatured conditions for immunoprecipitation

• Batch-to-batch variability:

  • Challenge: Polyclonal antibody preparations show variability between lots

  • Solution: Reserve single lots for complete experimental series; validate each new lot against standard samples; consider monoclonal alternatives for critical applications

• Matrix interference in complex samples:

  • Challenge: Serum, tissue homogenates, or ES products may contain interfering substances

  • Solution: Implement more stringent washing protocols; optimize blocking conditions (5% milk has proven effective) ; consider sample clean-up methods before analysis

• Quantification challenges:

  • Challenge: Accurately quantifying CPI-1 levels, especially for comparison across studies

  • Solution: Include recombinant protein standards at known concentrations; normalize to housekeeping proteins; utilize digital image analysis software for densitometry

These solutions have been validated across multiple studies and will enhance data quality and reproducibility in CPI-1 research.

How can CPI-1 antibodies be used to investigate its role in cell signaling pathways?

CPI-1 antibodies are powerful tools for dissecting signaling pathway involvement, particularly in the OXPHOS/MEK/ERK axis:

• Pathway activation/inhibition studies:

  • Use CPI-1 antibodies in combination with phospho-specific antibodies (p-MEK1/2, p-ERK1/2) to assess downstream signaling events

  • Implement time-course experiments (15min, 30min, 1h, 2h, 4h) following CPI-1 overexpression or inhibition

  • Combine with pathway-specific inhibitors (Rotenone for OXPHOS, PD98059 for MEK/ERK) to establish causality

• Protein-protein interaction network mapping:

  • Employ proximity ligation assays with CPI-1 antibodies and antibodies against suspected interaction partners

  • Perform sequential co-immunoprecipitation experiments to identify multi-protein complexes

  • Validate interactions through reciprocal pulldowns and mass spectrometry analysis

• Subcellular localization analysis:

  • Use immunofluorescence with organelle markers to track CPI-1 localization under different signaling conditions

  • Implement cellular fractionation followed by immunoblotting to quantify redistribution between compartments

  • Research has revealed CPI-1 interaction with mitochondrial components, particularly GRIM19 in complex I

• Functional readouts:

  • Correlate CPI-1 expression/localization with measurements of mitochondrial complex I activity

  • Assess ATP production rates in parallel with signaling pathway activation

  • Monitor cellular migration and invasion as downstream functional consequences

This multilayered approach has successfully demonstrated that CST1 enhances mitochondrial respiratory chain complex I activity to activate the OXPHOS/MEK/ERK axis in ESCC, revealing its oncogenic role .

What experimental approaches can determine if CPI-1 is a viable therapeutic target in disease models?

Evaluating CPI-1 as a therapeutic target requires a comprehensive experimental strategy spanning in vitro to in vivo approaches:

• Target validation studies:

  • Implement genetic approaches (siRNA, CRISPR-Cas9) to knockdown/knockout CPI-1 in disease models

  • Assess phenotypic consequences on cell migration, invasion, and viability in cancer cell lines

  • Analyze mitochondrial respiratory chain complex I enzyme activity and OXPHOS levels following manipulation

• Small molecule inhibitor screening:

  • Develop high-throughput fluorescence-based assays measuring protease inhibition

  • Screen chemical libraries for compounds that disrupt CPI-1 interaction with target proteases

  • Validate hits using secondary assays (thermal shift, microscale thermophoresis)

• In vivo efficacy assessment:

  • Establish xenograft models overexpressing CPI-1 (e.g., lenti-CST1 ESCC models)

  • Test intervention strategies including:

    • Direct CPI-1 inhibitors

    • Downstream pathway inhibitors (Rotenone for OXPHOS, PD98059 for MEK/ERK)

    • Combination approaches

  • Monitor tumor growth, metastasis formation, and survival metrics

• Pharmacological evaluation:

  • Determine pharmacokinetic parameters of lead compounds

  • Assess toxicity profile at therapeutic doses

  • Previous studies with cysteine protease inhibitors showed efficacy without significant toxicity at plasma concentrations between 5-19μM

• Biomarker development:

  • Use CPI-1 antibodies to identify patient populations likely to respond to therapy

  • Develop companion diagnostics for therapy stratification

  • Monitor treatment response using CPI-1 levels or activity

This approach has successfully demonstrated that CST1/OXPHOS represents a promising target for ESCC treatment and that cysteine protease inhibitors can effectively treat parasitic infections without significant host toxicity .

How can we design experiments to understand CPI-1's immunomodulatory functions in different disease contexts?

Designing experiments to elucidate CPI-1's immunomodulatory functions requires specialized approaches across multiple immune contexts:

• Macrophage polarization studies:

  • Isolate monocyte-derived macrophages (MDMs) and treat with recombinant CPI-1 (10μg/mL)

  • Analyze M1 markers (IL-12β, IL-6, IL-1β, iNOS) and M2 markers (CD206, CD163, CD301, Arg1, Ym-1, IL-10) via qPCR

  • Include TLR pathway analysis using specific activators (Pam3CSK4 for TLR2, LPS for TLR4) and blocking antibodies (TL2.1, 7E3)

  • Validate at protein level using flow cytometry for surface markers and ELISA for secreted cytokines

• T cell response modulation assessment:

  • Co-culture CPI-1-treated macrophages with CFSE-labeled CD4+ T cells

  • Measure proliferation by flow cytometry tracking CFSE dilution

  • Analyze T cell subset differentiation by measuring Th1 (T-bet, IFN-γ), Th2 (Gata-3, IL-4), Th17 (ROR-γ, IL-17A), and Treg (Foxp3, TGF-β) markers

  • Evaluate immune checkpoint activation through PD-L2/PD-1 and CTLA-4/CD80 expression

• Antigen presentation pathway analysis:

  • Measure MHC-II expression on antigen-presenting cells treated with CPI-1

  • Track antigen processing using fluorescent-labeled model antigens

  • Assess cathepsin activity in endolysosomal compartments using activity-based probes

  • Analyze cross-presentation capacity using OT-I/OT-II systems

• In vivo immune response models:

  • Administer recombinant CPI-1 in inflammatory disease models (autoimmunity, infection)

  • Monitor disease progression, inflammatory markers, and immune cell infiltration

  • Analyze tissue-specific immune responses through immunohistochemistry and cell isolation

  • Test therapeutic potential in relevant disease models

This experimental framework has revealed that BsCPI-1 induces a Th1/Th2 mixed immune response dominated by Th2 cells, promotes Treg differentiation, and activates immunosuppressive PD-1/PD-L2 and CTLA-4/CD80 pathways , providing a foundation for investigating similar functions across different disease contexts.

What are the emerging techniques for studying CPI-1 function that might overcome current limitations?

Several cutting-edge approaches are poised to transform CPI-1 research:

• CRISPR-based genetic screens:

  • Genome-wide CRISPR screens to identify synthetic lethal interactions with CPI-1 in cancer contexts

  • CRISPRi/CRISPRa for dose-dependent modulation of CPI-1 expression

  • Base editors for introducing specific mutations to dissect structure-function relationships

  • CRISPR knock-in approaches to tag endogenous CPI-1 for live-cell imaging

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize CPI-1 localization at nanoscale resolution

  • FRET/FLIM approaches to detect real-time protein-protein interactions with GRIM19 and complex I components

  • Intravital microscopy to track CPI-1 function in living tissues during disease progression

  • Expansion microscopy for enhanced visualization of subcellular compartments

• Proteomic approaches:

  • Proximity labeling (BioID, APEX) to comprehensively map CPI-1 interaction networks

  • Thermal proteome profiling to identify direct binding partners and off-targets

  • Cross-linking mass spectrometry to capture transient interactions

  • Phosphoproteomics to fully characterize signaling cascades activated by CPI-1

• Single-cell technologies:

  • scRNA-seq to characterize heterogeneous responses to CPI-1 in immune and cancer cells

  • CyTOF for high-dimensional protein expression analysis in response to CPI-1

  • Spatial transcriptomics to map CPI-1 expression and effects within tissue architecture

  • Microfluidic approaches for manipulating single cells expressing different levels of CPI-1

• Computational methods:

  • Molecular dynamics simulations to predict effects of mutations on CPI-1 function

  • AI-driven structure prediction to model CPI-1 interactions with novel partners

  • Systems biology approaches to integrate multi-omics data into comprehensive pathway models

  • Virtual screening to identify novel inhibitors targeting the CPI-1/GRIM19 interface

These emerging approaches will help resolve current limitations in understanding the mechanistic details of how CST1/CPI-1 enhances mitochondrial respiratory chain complex I activity and mediates downstream signaling events .

How might research on CPI-1 intersect with emerging concepts in mitochondrial regulation and immune evasion?

The intersection of CPI-1 research with emerging concepts in mitochondrial biology and immune regulation presents exciting new frontiers:

• Mitochondrial dynamics and metabolism:

  • Investigate how CPI-1/GRIM19 interaction affects mitochondrial fission/fusion events

  • Explore metabolic reprogramming beyond OXPHOS, including glutamine metabolism and one-carbon metabolism

  • Examine potential roles in mitophagy regulation and mitochondrial quality control

  • Research has established CPI-1's impact on complex I activity and OXPHOS , but broader mitochondrial functions remain unexplored

• Immunometabolism connections:

  • Study how CPI-1-mediated metabolic changes in tumor cells affect tumor-infiltrating immune cells

  • Investigate metabolic competition in the tumor microenvironment

  • Examine how CPI-1 expression affects response to immunotherapy

  • Explore connections between mitochondrial status and PD-L2/PD-1 pathway activation observed in CPI-1 research

• Extracellular vesicle biology:

  • Determine if CPI-1 is packaged into extracellular vesicles (EVs)

  • Study potential horizontal transfer of CPI-1 between cells via EVs

  • Investigate EV-mediated signaling in cancer progression and immune modulation

  • Parasitic CPIs are known to be secreted , suggesting potential EV involvement

• Integrated stress response:

  • Explore how CPI-1 affects cellular stress responses, particularly mitochondrial unfolded protein response

  • Investigate connections to cellular proteostasis networks

  • Examine potential roles in ER-mitochondria contact sites and calcium regulation

  • Study implications for cellular adaptations to environmental stressors

• Evolution of immune evasion strategies:

  • Compare CPI-1 functions across species from parasites to humans

  • Investigate convergent evolution of immune modulation mechanisms

  • Explore how host-pathogen interactions have shaped CPI-1 functions

  • Research shows similar immunomodulatory mechanisms between parasite CPIs and cancer-associated CST1

This integrative approach will reveal how CPI-1's diverse functions in mitochondrial regulation and immune modulation represent evolutionary adaptations that are exploited in disease contexts.

What standardized protocols and resources are available for researchers working with CPI-1 antibodies?

Researchers investigating CPI-1 can access several standardized resources and protocols:

• Antibody validation resources:

  • Commercially available CPI-1 antibodies with validation data

  • Recombinant proteins for positive controls (available from MyBioSource, Cusabio)

  • Pre-immune sera for negative controls in immunoassays

• Standardized protocols:

  • Western blot procedures optimized for CPI-1 detection (1:1000 dilution, nitrocellulose membranes, 5% milk blocking)

  • Immunofluorescence protocols for tracking CPI-1 localization

  • Co-immunoprecipitation methods for detecting protein-protein interactions

  • Enzymatic inhibition assays using fluorogenic substrates (Z-Phe-Arg-AMC, Z-Arg-Arg-AMC)

• Expression systems:

  • Bacterial expression systems for recombinant CPI-1 production (E. coli)

  • Mammalian expression vectors for cell-based assays

  • Viral delivery systems (lentivirus) for stable expression in experimental models

• Cell and animal models:

  • Cell lines with characterized CPI-1 expression profiles

  • siRNA and shRNA constructs for CPI-1 knockdown

  • Xenograft models for in vivo studies

  • TLR2/4 knockout systems for immunomodulation studies

• Analytical tools and databases:

  • Protease substrate databases for identifying potential targets

  • Web-based tools for analyzing protease-inhibitor interactions

  • Structural databases containing cystatin family proteins

  • Pathway analysis resources for interpreting signaling data

These resources provide researchers with validated starting points for investigating CPI-1 biology across diverse experimental systems and applications.

What alternative approaches can be used when CPI-1 antibodies fail to work in certain applications?

When CPI-1 antibodies present limitations, researchers can employ several alternative approaches:

• Genetic tagging strategies:

  • CRISPR knock-in of epitope tags (FLAG, HA, V5) to endogenous CPI-1

  • This allows detection with well-characterized commercial tag antibodies

  • Expression of fusion proteins (GFP, luciferase) for live monitoring

  • Use of split reporter systems (BiFC) to visualize protein interactions

• Functional surrogates:

  • Measure enzymatic activity of target proteases (cathepsins B/L, papain) as indirect readout of CPI-1 function

  • Assess OXPHOS activity via Seahorse analyzer as downstream indicator

  • Monitor MEK/ERK pathway activation through phosphorylation status of pathway components

  • Track cellular phenotypes (migration, invasion) as functional consequences

• Nucleic acid detection:

  • RT-qPCR to quantify CPI-1 mRNA expression

  • RNA-FISH for spatiotemporal visualization of transcript localization

  • Single-cell transcriptomics for heterogeneity assessment

  • These approaches circumvent protein detection challenges entirely

• Activity-based protein profiling:

  • Use biotinylated or fluorescent activity-based probes that bind active site of proteases

  • Measure protease protection by CPI-1 through reduced probe binding

  • Applicable in complex biological samples without requiring antibodies

• Mass spectrometry approaches:

  • Targeted proteomics (MRM/PRM) for absolute quantification of CPI-1

  • Global proteomics to detect changes in CPI-1 interaction networks

  • Cross-linking mass spectrometry to capture transient protein interactions

  • CETSA (Cellular Thermal Shift Assay) to detect target engagement