Proteolytic Enzyme Networks in Cell Injury: Activation Sequence, Substrate Cleavage, and Experimental Attribution

The proteolytic enzyme network in cell injury involves calpains, caspases, cathepsins, MMP/ADAM family proteases, and the ubiquitin-proteasome system. Different proteolytic enzymes correspond to Ca²⁺ overload, apoptotic execution, lysosomal injury, extracellular matrix remodeling, and disrupted protein homeostasis. Experimental design should focus on establishing an evidence chain based on the detection window, substrate cleavage fragments, inhibitor specificity, and result attribution.

Keywords: cell injury; proteolytic enzyme; calpain; caspase; cathepsin; MMP; proteasome; substrate cleavage

1 Research Positioning of the Proteolytic Enzyme Network

1.1 Network Composition and Interpretation Framework

(1) Functional division

Calpains primarily mediate Ca²⁺-dependent limited proteolysis and frequently act on cytoskeletal proteins, membrane-associated structures, and adhesion complexes. Caspases are more closely associated with apoptotic execution and substrate cleavage during inflammatory cell death. Once released from lysosomes, cathepsins can amplify mitochondrial injury or inflammasome signaling. MMPs and ADAMs mainly participate in extracellular matrix degradation and membrane protein shedding. The ubiquitin-proteasome system maintains protein homeostasis by degrading ubiquitinated proteins.

(2) Temporal sequence

Proteolytic events show clear stage specificity. Increased Ca²⁺ levels and calpain activation usually occur during the early phase of injury. Caspase cascades often indicate the apoptotic execution phase. Cytosolic release of cathepsins suggests lysosomal membrane permeabilization. Enhanced MMP activity is often associated with tissue barrier disruption, migration and invasion, or remodeling of the inflammatory microenvironment. Single endpoint detection can easily miss critical activation windows.

(3) Spatial localization

Different proteolytic enzymes act in different subcellular regions. Calpains are mainly localized in the cytoplasm, membrane cytoskeleton, and local Ca²⁺ microdomains. Caspases primarily cleave substrates in the cytoplasm and nucleus. Cathepsin analysis should focus on lysosomal localization and cytosolic release. MMPs and ADAMs often act on the extracellular matrix and membrane-proximal regions. Proteasomes are distributed in the cytoplasm and nucleus. Mechanistic attribution should therefore be combined with subcellular fractionation, immunofluorescence, or activity probe results.

Table 1 Major Proteolytic Enzyme Systems in Cell Injury

Proteolytic Enzyme System	Main Activation Conditions	Typical Action Site	Common Substrates or Readouts	Injury Interpretation
Calpain	Ca²⁺ overload, membrane injury, excitotoxicity	Cytoplasm, membrane cytoskeleton, adhesion structures	αII-spectrin, talin, FAK cleavage	Ca²⁺-dependent limited proteolysis
Caspase	Mitochondrial pathway, death receptor pathway, inflammasome	Cytoplasm, nucleus	Caspase-3, PARP, lamin cleavage	Apoptotic or inflammatory cell death execution phase
Cathepsin	Lysosomal membrane permeabilization, acidic organelle injury	Lysosome, cytoplasm	Cathepsin release, Bid cleavage	Lysosomal injury and amplification of death signaling
MMP/ADAM	Inflammatory cytokines, ECM remodeling, membrane protein shedding	Extracellular matrix, membrane-proximal regions	Gelatinase activity, ECM degradation, receptor shedding	Matrix disruption, barrier injury, migration and invasion
Proteasome	Ubiquitinated protein accumulation, protein quality control stress	Cytoplasm, nucleus	Changes in ubiquitinated proteins, p53, IκBα	Protein homeostasis imbalance and stress regulation

2 Key Proteolytic Enzyme Systems and Injury Attribution

2.1 Calpain Pathway

(1) Ca²⁺-dependent activation

Calpains are typical Ca²⁺-dependent cysteine proteases involved in cell injury. Ischemia/hypoxia, excitotoxicity, oxidative stress, mechanical injury, and mitochondrial dysfunction can all induce intracellular Ca²⁺ elevation and promote calpain activation.

(2) Substrate cleavage characteristics

Calpains usually mediate limited proteolysis rather than complete substrate degradation. Characteristic cleavage of cytoskeletal or adhesion-related proteins such as αII-spectrin, talin, FAK, and paxillin often suggests the involvement of Ca²⁺-dependent structural injury. If calpain inhibition reduces both substrate cleavage and cell injury readouts, this further supports a functional role for calpain.

2.2 Caspase Pathway

(1) Apoptotic execution

Caspase-3, caspase-6, and caspase-7 are important proteases in the apoptotic execution phase and can cleave substrates such as PARP, lamin, and ICAD. Increased cleaved caspase-3 and cleaved PARP are commonly used to assess apoptotic execution, but they do not independently explain the upstream initiation mechanism.

(2) Cell death mode switching

Caspase-8 connects the death receptor pathway, apoptosis, and necroptosis regulation. If z-VAD-FMK reduces PARP cleavage but does not improve cell death, p-MLKL, GSDMD cleavage, and LDH release should be further examined to determine whether a switch in cell death mode has occurred.

2.3 Cathepsin Pathway

(1) Lysosomal membrane permeabilization

Cathepsins B, D, and L are normally localized inside lysosomes. Oxidative stress, lipid peroxidation, drug stimulation, and uptake of crystals or particles can induce lysosomal membrane permeabilization, causing cathepsins to be released into the cytoplasm. Cytosolic cathepsins can promote Bid cleavage, mitochondrial injury, and amplification of the caspase cascade.

(2) Connection with inflammatory signaling

Cathepsin B release is frequently associated with NLRP3 inflammasome activation. If cytosolic cathepsin release, caspase-1 activation, GSDMD cleavage, and IL-1β maturation are observed simultaneously, lysosomal injury-mediated inflammatory cell death should be considered.

2.4 MMP/ADAM Pathway

(1) Matrix degradation

MMP-2 and MMP-9 are commonly used to evaluate gelatin-degrading activity. MMP-3 and MMP-13 are associated with tissue remodeling, cartilage destruction, and inflammatory injury. Increased MMP activity more often indicates extracellular matrix remodeling, barrier disruption, or enhanced migration and invasion, and should not be directly equated with increased cell death.

(2) Membrane protein shedding

ADAM family proteases and some MMPs can cleave extracellular domains of membrane proteins, affecting receptor signaling, adhesion molecules, and inflammatory factor release. When studying cell junction disruption, epithelial barrier injury, or receptor shedding, soluble cleavage fragments, reduced membrane protein levels, and responses to MMP/ADAM inhibitors should be evaluated together.

2.5 Ubiquitin-Proteasome System

(1) Regulation of protein homeostasis

The proteasome maintains protein quality control by degrading ubiquitinated proteins. Oxidative stress, ER stress, and mitochondrial injury can increase the burden of misfolded proteins. When proteasome function is impaired, accumulation of ubiquitinated proteins can further induce stress responses, inflammatory signaling, and cell death.

(2) Interpretation boundaries of inhibitors

Proteasome inhibitors such as MG132 can be used to study target protein stability, but their effects are broad and may induce ER stress, ROS elevation, and apoptosis. When studying proteasome-related mechanisms, attribution should combine ubiquitinated protein accumulation, proteasome activity, CHX chase assays, and cytotoxicity indicators.

Table 2 Experimental Attribution Points for Proteolytic Enzyme Systems

Pathway	Key Evidence	Recommended Validation	Common Misinterpretation
Calpain	Ca²⁺ elevation, increased calpain activity, αII-spectrin cleavage	EGTA, calpeptin, substrate cleavage detection	Inferring calpain activation solely from Ca²⁺ elevation
Caspase	Cleaved caspase-3, PARP cleavage, Annexin V positivity	z-VAD-FMK, caspase activity assay	Inferring the entire apoptotic mechanism solely from PARP cleavage
Cathepsin	Lysosomal membrane permeabilization, cytosolic cathepsin release	Fractionation markers, cathepsin inhibitors, activity probes	False positives caused by fractionation contamination
MMP/ADAM	Gelatinase activity, ECM degradation, membrane protein cleavage fragments	MMP inhibitors, migration/invasion assays, cell viability controls	Directly interpreting matrix remodeling as cell death
Proteasome	Ubiquitinated protein accumulation, altered proteasome activity	MG132, CHX chase, target protein half-life	Attributing all MG132 effects to target protein stability

3 Detection Strategies for Proteolytic Enzyme Networks

3.1 Enzyme Activity Detection

Enzyme activity assays are suitable for evaluating the current functional state of proteases. Calpains, caspases, cathepsins, MMPs, and proteasomes can all be detected using substrate-based assays or activity probes. Experimental design should consider lysis buffer composition, pH, chelators, detergents, sample autofluorescence, and substrate specificity. Enzyme activity readouts are suitable for screening and time-course analysis, but they cannot replace substrate cleavage detection and functional phenotyping.

3.2 Substrate Cleavage Detection

Substrate cleavage is more directly related to functional proteolysis than total protein expression. Calpain activation can be evaluated by cleavage of αII-spectrin, talin, or FAK. Caspase activity can be assessed by PARP, lamin, and caspase self-cleavage. Cathepsin involvement can be evaluated by Bid cleavage and cytosolic release. MMP activity can be examined using gelatin substrates or ECM degradation. Proteasome activity can be inferred from ubiquitinated protein accumulation and target protein stability. Substrate cleavage fragments should be attributed based on molecular weight, cleavage site, and reversal by inhibitors.

3.3 Inhibitors and Genetic Interventions

Inhibitors can be used to verify the extent of protease involvement, but their specificity is often limited. Calpain inhibitors may affect other cysteine proteases. z-VAD-FMK may alter cell death mode. MG132 can induce broad cellular stress. Key conclusions should be supported by inhibitors of different structural classes, siRNA/CRISPR intervention, overexpression of endogenous inhibitors, or rescue experiments.

Table 3 Experimental Design Framework for Proteolytic Enzyme Networks

Experimental Question	Recommended Detection Combination	Key Controls	Result Interpretation
Is Ca²⁺-dependent injury present?	Ca²⁺ imaging, calpain activity, αII-spectrin cleavage	EGTA, calpain inhibitor	Determine whether calpain is an early injury node
Has apoptosis entered the execution phase?	Cleaved caspase-3, PARP cleavage, Annexin V	z-VAD-FMK, negative control	Determine the degree of caspase cascade involvement
Is lysosomal injury involved?	LysoTracker, cathepsin release, cathepsin activity	Cathepsin inhibitor, fractionation markers	Determine the connection between LMP and death signaling
Is matrix degradation enhanced?	MMP activity, ECM substrate changes, migration/invasion assays	MMP inhibitor, cell viability control	Distinguish matrix remodeling from cytotoxicity
Is protein homeostasis disrupted?	Ubiquitinated proteins, proteasome activity, ER stress indicators	MG132, CHX chase	Determine mechanisms of protein degradation or accumulation
Does a drug act on the proteolytic network?	Enzyme activity, cleavage fragments, cell death, inhibitor combination	Vehicle control, multi-pathway inhibitors	Distinguish direct inhibition from secondary protection

4 Application Logic in Common Injury Models

4.1 Ischemia/Hypoxia and Reperfusion Injury

In ischemia-reperfusion models, Ca²⁺ overload, increased ROS, and decreased mitochondrial membrane potential often occur together. Calpain activation can serve as an early indicator of structural injury, caspase cascades reflect apoptotic execution, and cathepsin release suggests lysosomal injury involvement. Experiments should sequentially detect Ca²⁺ changes, calpain activity, substrate cleavage, mitochondrial indicators, and cell death.

4.2 Neuronal Injury

In excitotoxicity and neurodegenerative injury, calpain often contributes to axonal cytoskeletal cleavage, synaptic protein alterations, and membrane structural damage. If calpain inhibition reduces αII-spectrin cleavage and improves axonal integrity or neuronal survival, this supports its functional role in early injury. If caspase-3 activation also occurs, further analysis is needed to determine whether calpain lies upstream of caspase activation.

4.3 Lysosomal Injury and Inflammatory Models

Particles, crystals, lipid peroxidation, and certain drug stimuli can induce lysosomal membrane permeabilization. In these models, cytosolic cathepsin release, NLRP3 activation, caspase-1 activation, GSDMD cleavage, and IL-1β maturation should be detected together. If cathepsin inhibitors reduce inflammasome indicators, this supports the involvement of lysosomal injury in inflammatory cell death.

4.4 Tumor Migration and Tissue Remodeling Models

In models of migration and invasion, fibrosis, inflammatory tissue destruction, and barrier injury, the MMP/ADAM pathway has greater interpretive value. MMP activity, ECM degradation, membrane protein cleavage fragments, cell migration ability, and cell viability should be analyzed separately to avoid misinterpreting reduced migration caused by cytotoxicity as specific inhibition of the MMP pathway.

Table 4 Proteolytic Enzyme Combinations in Different Injury Models

Model Type	Priority Pathways	Recommended Indicators	Interpretation Focus
Ischemia-reperfusion	Calpain, caspase, cathepsin	Ca²⁺, calpain activity, PARP cleavage, cathepsin release	Determine the sequence of early structural injury and death execution
Excitotoxicity	Calpain, caspase	αII-spectrin cleavage, caspase-3, neuronal survival	Distinguish Ca²⁺-dependent cleavage from apoptotic execution
Lysosomal injury	Cathepsin, caspase-1	Cathepsin B release, GSDMD, IL-1β	Determine the connection between LMP and inflammasome activation
Tumor migration and invasion	MMP/ADAM, calpain	MMP-2/9 activity, talin/FAK cleavage, migration assays	Distinguish matrix remodeling from cytotoxicity
Protein homeostasis stress	Proteasome, caspase	Ubiquitinated proteins, ER stress, cleaved caspase-3	Determine the relationship among protein accumulation, stress, and death

5 Abnormal Results and Attribution Control

5.1 Increased Enzyme Activity Without Obvious Substrate Cleavage

This pattern is often caused by a mismatched sampling window, unsuitable substrate selection, insufficient specificity of the enzyme activity substrate, or instability of cleavage fragments. A time-course experiment, a more representative substrate cleavage antibody, and inhibitor reversal experiments are recommended.

5.2 Enhanced Substrate Cleavage Without Inhibitor Reversal

The substrate may be cleaved by multiple proteases. If calpain inhibitors fail to reverse αII-spectrin cleavage, caspase inhibitor controls should be added. If cathepsin inhibitors fail to reverse Bid cleavage, the mitochondrial pathway and caspase cascade should be examined. A negative result with a single inhibitor does not directly exclude involvement of the proteolytic network.

5.3 Inhibitor Improves Cell Viability Without Altering Enzyme Activity

The inhibitor may act on non-target pathways, or the detection window may not coincide with the peak activity of the target enzyme. Sampling time should be reset, and substrate cleavage fragments, upstream stimuli, and cellular phenotypes should be examined. If an inhibitor only improves cell viability without enzyme activity or substrate-level evidence, the protective effect should not be directly attributed to the target protease.

Table 5 Common Abnormal Results and Optimization Strategies in Proteolytic Enzyme Network Studies

Abnormal Finding	Possible Cause	Optimization Strategy
Enzyme activity signal increases but substrate cleavage is weak	Mismatched time window or unsuitable substrate selection	Set a time course and replace substrate cleavage indicators
Substrate cleavage increases but inhibitor is ineffective	Shared cleavage by multiple proteases or insufficient inhibitor dose	Add inhibitors for other pathways and perform dose gradients
z-VAD-FMK reduces PARP cleavage but does not reduce death	Cell death mode switching or compensation by non-apoptotic death	Detect p-MLKL, GSDMD, and LDH release
Abnormally increased cytosolic cathepsin signal	Fractionation contamination or overly harsh lysosomal disruption during processing	Add fractionation markers and validate by immunofluorescence
MMP activity increases but cell number decreases	Cytotoxicity interferes with migration or secretion readouts	Detect cell viability and LDH release simultaneously
Multiple pathways are activated after MG132 treatment	Proteasome inhibition induces broad cellular stress	Reduce dose, shorten treatment duration, and combine with CHX chase

6 Selection of Related Products and Materials

6.1 Calpain Pathway-Related Products

Product Category	Cat. No.	Product/Material Name	Grade & Purity	Application Positioning
Enzyme source	C1441522	Calpain-1 (pig)		Used for in vitro calpain-1 enzyme activity systems, substrate cleavage reactions, and inhibitor screening model establishment
Enzyme source	np001116	Calpain-1 from Human Erythrocyte	Native,EnzymoPure™,≥95%(SDS-PAGE),≥1000 U/mg protein; Protein concentration: See COA	Suitable as a human-derived calpain-1 positive enzyme source for Ca²⁺-dependent proteolysis systems and inhibitor effect validation
Enzyme source	np001115	Calpain-1, Porcine Erythrocytes		Used for porcine calpain-1 enzyme source systems, in vitro substrate cleavage, and optimization of calpain reaction conditions
Enzyme source	C755293	Calpain-1, Porcine Erythrocytes	Calpain-1, Porcine Erythrocytes, is a native calpain-1. A heterodimeric cysteine proteinase with low Ca2+ requirement (EC₅₀ = 2 µM).	Suitable for low-Ca²⁺ requirement calpain-1 activity reactions, substrate cleavage, and inhibitor effect comparison
Substrate	C1453940	Calpain-1 substrate, fluorogenic		Used for calpain-1 fluorogenic substrate activity assays
Substrate	C1443233	Calpain substrate		Used for calpain substrate cleavage and enzyme activity readout establishment
Inhibitor	A275929	ALLM	≥95%	Used for calpain inhibition validation and attribution of Ca²⁺-dependent injury
Inhibitor	A274819	ALLN	≥95%	Used for validating calpain involvement and inhibitor controls
Inhibitor	A420582	ALLN	10mM in DMSO	Suitable as a ready-to-use calpain inhibitor stock for cell treatment or pharmacological screening
Inhibitor	C695638	Calpain inhibitor II	≥95%	Used for calpain inhibition experiments and can be combined with substrate cleavage detection
Inhibitor	P276048	PD 151746	≥95%	Suitable for calpain-specific intervention and pharmacological screening experiments
Inhibitor	L275900	Z-LLY-FMK	≥95%	Used for inhibitory validation of calpain-mediated substrate cleavage
Inhibitor	C420784	Calpeptin	Moligand™, 10mM in DMSO	Suitable for cell-permeable calpain inhibition experiments
Inhibitor	C274691	Calpeptin	Moligand™, ≥95%	Used for calpain inhibition validation in cellular models and intervention in Ca²⁺-dependent injury
Inhibitor	N276203	NYC-488	≥94%	Used for calpain inhibitor comparison and pharmacological screening
Inhibitor	C336446	Calpain Inhibitor VI	≥95%	Used for calpain inhibition validation and can be paired with calpain activity readouts and substrate cleavage detection
Inhibitor	C341237	Calpain Inhibitor XII	≥90%	Used for calpain pathway inhibition and cross-validation with different inhibitors
Inhibitor	C1436419	Calpain Inhibitor-2		Used for inhibition experiments of calpain-mediated proteolysis
Inhibitor	C1419148	Calpain inhibitor V		Used for combined validation of calpain inhibitors and intervention in injury models
Inhibitor	C1497109	Calpain-2-IN-1	Moligand™, 10 mM in DMSO	Used for calpain-2-targeted inhibition and cell treatment experiments
Inhibitor	C649440	Calpain-2-IN-1	≥98%	Used for calpain-2 inhibitor validation and CAPN2-related mechanistic studies
Inhibitor	M611796	mercaptoacrylate inhibitor of calpain 1	Moligand™	Used for calpain-1 inhibition and isoform-related pharmacological validation
Antibody	Ab092523	Recombinant Calpain 1 Antibody	ExactAb™, Validated, Recombinant, 2.0 mg/mL	Used for calpain-1 protein expression detection, WB validation, and analysis of calpain-1-related injury pathways
Antibody	Ab327205	Recombinant Calpain 1 Antibody	KD Validation	Used for calpain-1 expression detection and CAPN1 signal validation in knockdown models
Antibody	Ab092535	Calpain 2 Antibody	Carrier Free, ExactAb™, Validated, High Performance, See COA	Used for calpain-2 protein expression detection, CAPN2 pathway validation in injury models, and isoform distinction
Antibody	Ab327201	Recombinant Calpain 2 Antibody	KD Validation	Used for calpain-2 expression detection and CAPN2 signal validation in knockdown models
Antibody	Ab092526	Calpain 10 Antibody	See COA	Used for calpain 10 protein level detection and calpain isoform analysis in specific cellular models
ELISA kit	EJ1512394	Rat Calpain 1 (Calpain 1) ELISA Kit	BioReagent	Used for detecting calpain 1 levels in rat cell, tissue, or body fluid samples
ELISA kit	EJ1512395	Rat Calpain 2 (Calpain 2) ELISA Kit	BioReagent	Used for detecting calpain 2 levels in rat samples and evaluating CAPN2 changes in injury models
ELISA kit	EJ1515072	Human Calpain 2 (CAPN2) ELISA Kit	BioReagent	Used for detecting CAPN2 levels in human samples and suitable for cell injury and clinically related sample analysis

6.2 Caspase Pathway-Related Products

Product Category	Cat. No.	Product/Material Name	Grade & Purity	Application Positioning
Activity assay kit	C1372489	Caspase 3/7 Activity Assay Kit	BioReagent	Used for detecting caspase-3/7 executioner apoptotic activity
Live-cell assay kit	L1520214	Live Cell Caspase-3/7 Activity and Annexin V Dual Apoptosis Detection Kit (LumiDye™ 488 Caspase-3/7, LumiDye™ 594-Annexin V, Hoechst 33342)	BioReagent,sterile,for microscopy,Biological Stain,for fluorescence analysis	Used for simultaneous live-cell detection of caspase-3/7 activity and early apoptotic membrane externalization
Live-cell assay kit	L1520218	Live Cell Caspase-3/7 Activity and Annexin V Dual Apoptosis Detection Kit (LumiDye™ 488 Caspase-3/7, LumiDye™ 647-Annexin V, EthD Gold, Hoechst 33342)	BioReagent,sterile,for microscopy,Biological Stain,for fluorescence analysis	Suitable for combined analysis of live-cell apoptotic progression and membrane integrity
Inhibitor	Z421947	Z-VAD(OH)-FMK (Caspase Inhibitor VI)	10mM in DMSO	Used for broad-spectrum caspase inhibition and validation of cell death mode switching
Inhibitor	Q275003	QVD-OPh	≥95%, mixture of isomers	Used for intervention in caspase-dependent cell death
Inhibitor	M274709	Caspase-3/7 Inhibitor	≥97%	Used for validating caspase-3/7-specific inhibition
Antibody	Ab213595	Caspase 3 Mouse mAb	Carrier Free,ExactAb™,Azide Free,Validated,High Performance,PBS Only,≥95%(SDS-PAGE),1.0 mg/mL	Used for caspase-3 protein expression and apoptotic pathway detection
Antibody	Ab326059	Cleaved Caspase 8 Antibody	KD Validation	Used for validating caspase-8 activation and the death receptor pathway
Antibody	Ab326939	Recombinant cleaved Caspase-9 Antibody	KD Validation	Used for detecting caspase-9 activation in the mitochondrial apoptotic pathway

6.3 Cathepsin and Lysosomal Injury-Related Products

Product Category	Cat. No.	Product/Material Name	Grade & Purity	Application Positioning
Antibody	Ab093155	Cathepsin B Mouse mAb	Carrier Free, ExactAb™, Azide Free, Validated, High Performance, See COA	Used for cathepsin B expression, localization, and lysosomal injury-related detection
Antibody	Ab215111	Cathepsin D Mouse mAb	Carrier Free,ExactAb™,Azide Free,Validated,High Performance,PBS Only,≥95%(SDS-PAGE),1.0 mg/mL	Used for cathepsin D expression and lysosomal pathway validation
Antibody	Ab093200	Cathepsin L/MEP Antibody	ExactAb™, Validated, 1.0 mg/mL	Used for detecting cathepsin L-related proteolytic pathways
Substrate	C1441538	Cathepsin D/E Substrate, Fluorogenic		Used for cathepsin D/E fluorogenic substrate activity assays
Substrate	C659428	Cathepsin D and E FRET Substrate acetate	≥98%	Used for cathepsin D/E FRET substrate-based activity assays
Inhibitor	E109034	E-64	Moligand™, ≥99%, protease inhibitor	Used for cathepsin/cysteine protease background control
Inhibitor	E123225	E-64d	Moligand™,≥98%,protease inhibitor	Suitable for cellular cathepsin B/L inhibition experiments
Inhibitor	C408095	Cathepsin Inhibitor 1	10mM in DMSO	Used for validating cathepsin pathway inhibition
Inhibitor	C1494949	Cathepsin L-IN-2	Moligand™, 10 mM in DMSO	Used for validating cathepsin L-related injury mechanisms
ELISA kit	EJ1514552	Human Cathepsin B (CTSB) ELISA Kit	BioReagent	Used for detecting CTSB levels in human samples
ELISA kit	EJ1514554	Human Cathepsin D (Cath-D) ELISA Kit	BioReagent	Used for detecting cathepsin D levels in human samples

6.4 MMP/ADAM Pathway-Related Products

Product Category	Cat. No.	Product/Material Name	Grade & Purity	Application Positioning
Substrate	M1442064	MMP-1/MMP-9 Substrate, Fluorogenic		Used for MMP substrate cleavage and gelatinase-related activity detection
Inhibitor	M276513	MMP-2/MMP-9 Inhibitor I	Moligand™,≥99%	Used for validating MMP-2/9-dependent matrix degradation
Inhibitor	M338143	MMP-2/MMP-9 Inhibitor II	≥95%	Used for MMP-2/9 activity inhibition and migration/invasion mechanism analysis
Inhibitor	M1494998	MMP-2/MMP-9-IN-1	Moligand™, 10 mM in DMSO	Suitable for MMP-2/9 inhibition experiments in cell models
Inhibitor	M412536	MMP-9-IN-1	≥98%	Used for MMP-9-related inflammatory injury, ECM degradation, and migration assays
Inhibitor	M274731	MMP-9 inhibitor	≥95%	Used for MMP-9 pathway intervention and matrix degradation attribution
Antibody	Ab115657	MMP2 Mouse mAb	Carrier Free, ExactAb™, Validated, High Performance, See COA	Used for detecting MMP-2 protein expression and secretion levels
Antibody	Ab115721	MMP9 antibody	ExactAb™, Validated, Recombinant, See COA	Used for validating MMP-9 expression and inflammation/matrix remodeling
ELISA kit	EJ1514021	Human Matrix MetalloProteinase 2 (MMP-2) ELISA Kit	BioReagent	Used for detecting MMP-2 levels in human supernatants or samples
ELISA kit	EJ1514028	Human Matrix MetalloProteinase 9 (MMP-9) ELISA Kit	BioReagent	Used for detecting MMP-9 secretion levels and evaluating tissue injury
ELISA kit	EJ1514938	Human A Disintegrin And Metalloprotease 10 (ADAM10) ELISA Kit	BioReagent	Used for analyzing ADAM10-related membrane protein shedding pathways
ELISA kit	EJ1514939	Human A Disintegrin And Metalloprotease 17 (ADAM17) ELISA Kit	BioReagent	Used for studies of ADAM17-mediated receptor shedding and inflammatory factor release
Inhibitor	G169107	GI254023X	Moligand™, ≥98%(HPLC)	Used for validating ADAM10-dependent membrane protein shedding
Substrate	A1442055	ADAM-17 Substrate		Used for ADAM17 substrate cleavage activity detection

6.5 Proteasome and Sample Processing-Related Products

Product Category	Cat. No.	Product/Material Name	Grade & Purity	Application Positioning
Proteasome inhibitor	M126521	MG-132	Moligand™, ≥98%	Used for ubiquitinated protein accumulation, target protein stability, and proteasome inhibition experiments
Proteasome inhibitor	R166528	(R)-MG132	≥98%	Used for 20S proteasome inhibition and protein homeostasis stress models
Proteasome inhibitor	M275487	MG-115	Moligand™, ≥95%	Used for proteasome inhibition controls and target protein stability analysis
Proteasome inhibitor	D127401	CEP-18770 (Delanzomib)	≥97%	Used for studies of proteasome inhibition and cellular stress/apoptosis
Proteasome inhibitor	O129260	Oprozomib (ONX 0912)	Moligand™, ≥98%	Used for inhibition studies of 20S proteasome β5/LMP7-related activity
Proteasome detection	EJ1514821	Human Proteasome Subunit Alpha Type 5 (PSMα5) ELISA Kit	BioReagent	Used for detecting proteasome subunit expression levels
Proteasome detection	EJ1514824	Human Proteasome Activator Subunit 3 (PSME3/PA28γ) ELISA Kit	BioReagent	Used for detecting proteasome regulatory factors
Sample processing	P301902	Protease Inhibitor Cocktail	100×,EDTA free	Suitable for protecting protein samples when metal ion chelation interference should be avoided
Sample processing	P665818	Protease Inhibitor Cocktail	BioReagent, EDTA free, 100X	Suitable for protecting non-target proteins in cell/tissue lysate samples
Sample processing	P775091	Protease Inhibitor Cocktail (Suitable for mammalian cell and tissue extract, EDTA Free, 100X)	BioReagent, Suitable for mammalian cell and tissue extract, 100X, EDTA free	Suitable for processing mammalian cell and tissue samples
Sample processing	P752090	Protease and Phosphatase Inhibitor Cocktail (100×, EDTA-Free)	BioReagent, for western blot, for protein analysis, 100×	Suitable for WB samples in which both proteolysis and phosphorylation signals are evaluated
Sample processing	C1491689	Column Tissue&Cell Protein Extraction Kit (with Protease Inhibitor Cocktail)	BioReagent, for western blot, for protein analysis	Used for total protein extraction from cells or tissues and WB sample preparation

7 Frequently Asked Questions

7.1 How should the detection window for proteolytic enzymes be set in injury models?

The detection window should be established according to the kinetics of the injury model rather than selecting a fixed endpoint. Ca²⁺ imaging and calpain activity are more suitable for early time points. Caspase-3/PARP cleavage is suitable for the apoptotic execution phase. Cytosolic cathepsin release should be detected in parallel with lysosomal membrane permeabilization. MMP activity and ECM degradation usually require a longer observation period. A small-scale time-course experiment is recommended before determining sampling points for formal experiments.

7.2 How can αII-spectrin cleavage fragments be distinguished as calpain- or caspase-derived?

αII-spectrin can be cleaved by both calpain and caspase. Attribution should combine the molecular weight of cleavage fragments, timing of appearance, and inhibitor reversal results. If a calpain inhibitor markedly reduces a specific cleavage fragment while a caspase inhibitor has a weaker effect, this supports a calpain-derived fragment. If cleaved caspase-3 and PARP cleavage are also increased, caspase involvement should be considered.

7.3 How should subcellular fractionation contamination be controlled when detecting cytosolic cathepsin release?

Cytosolic cathepsin detection should include cytosolic, lysosomal, and mitochondrial compartment markers to confirm fractionation purity. If obvious lysosomal marker contamination appears in the cytosolic fraction, conclusions about cathepsin release should be interpreted cautiously. Cross-validation with immunofluorescence localization, lysosomal membrane permeabilization indicators, and cathepsin activity probes is recommended.

7.4 How can the dominant pathway be determined when multiple proteolytic enzymes are activated simultaneously?

The dominant pathway should be determined by integrating temporal sequence, substrate cleavage profile, magnitude of inhibitor reversal, and improvement in cellular phenotype. If calpain activation occurs earliest and its inhibition reduces downstream caspase cleavage and cell injury, calpain may be positioned upstream. If caspase inhibitors only reduce PARP cleavage but do not improve membrane rupture, caspases are not the only execution mechanism.

7.5 How can a mechanistic evidence chain be established when inhibitor specificity is insufficient?

A multilayer evidence strategy should be used. First, enzyme activity assays should confirm changes in the target protease activity. Second, characteristic substrate cleavage fragments should be detected. Third, inhibitors with different structural classes or genetic interventions should be used for cross-validation. Fourth, phenotypes such as cell death, migration, inflammation, or barrier function should be assessed for synchronized improvement. Fifth, nonspecific effects of inhibitors on other proteases and cell viability should be excluded. Mechanistic attribution is more reliable when multiple layers of evidence point in the same direction.

Studies of proteolytic enzymes in cell injury should avoid attribution based on a single indicator. Integrated analysis of enzyme activity, substrate cleavage, inhibitor response, subcellular localization, and cellular functional readouts is required to more accurately distinguish early injury nodes, execution-phase cleavage events, and secondary changes in protein homeostasis.

For more related articles, please see below:

[1] Construction and Evaluation Strategy of a Gentamicin-Induced Cell Injury Model