S100A6 Murine

What is S100A6 and what are its primary functions in murine systems?

S100A6 (also called calcyclin) is a low molecular weight Ca²⁺-binding protein belonging to the S100 protein family. In murine systems, S100A6 performs multiple critical functions:

• Regulation of hematopoietic stem cell (HSC) self-renewal through calcium buffering mechanisms

• Modulation of intracellular and mitochondrial calcium levels upon cytokine stimulation

• Regulation of actin filament dynamics through direct interaction with G-actin and F-actin

• Interaction with tropomyosin, particularly the Tpm1.8 isoform, to potentially regulate microfilament organization

• Governance of the Akt activation pathway, impacting mitochondrial metabolic function

When designing experiments involving S100A6, it's important to consider its calcium-dependent nature and its role in multiple cellular processes that may impact experimental outcomes.

How is S100A6 expression regulated in murine tissues?

S100A6 expression in murine models is:

• Cell-specific, with particularly high expression in mouse stomach and certain fibroblast populations

• Can be induced by multiple agents, particularly stress factors

• Primarily expressed in fibroblasts and epithelial cells

• Subject to complex regulatory mechanisms that control tissue-specific distribution

For accurate experimental design, researchers should account for this differential expression when selecting appropriate murine tissues for S100A6 studies and consider baseline expression levels when analyzing experimental results.

What phenotypic changes are observed in S100A6-deficient mice?

S100A6 knockout (S100A6KO) mice exhibit several distinctive phenotypic changes:

• Reduced total bone marrow cellularity

• Lower myeloid lineage output in peripheral blood

• Increased apoptotic cells in the long-term hematopoietic stem cell (LT-HSC) compartment

• Significantly reduced number of LT-HSCs and multipotent progenitor (MPP) cells

• Alterations in metabolic function, particularly in mitochondrial respiration

• Reduced hematopoietic colony-forming activity that can be rescued by Akt pathway activation

When working with S100A6KO mice, these baseline phenotypic changes must be considered when interpreting results from additional experimental manipulations.

What are reliable methods for detecting S100A6 expression in murine samples?

Several methodological approaches can be used to reliably detect S100A6 in murine samples:

• Fluorescence-activated cell sorting (FACS) for cell population analysis, particularly using LSKCD150⁺CD48⁻ markers for hematopoietic stem cell compartments

• Transcriptomic analysis using RNA sequencing, which has identified S100A6 as significantly downregulated in knockout models

• Proximity ligation assay, which has been used to demonstrate S100A6 complex formation with actin and tropomyosin in NIH3T3 fibroblasts

• Immunohistochemistry to visualize tissue-specific expression patterns

• Biochemical approaches using purified proteins to study direct interactions

For optimal results, combining multiple detection methods is recommended, particularly when studying subtle changes in S100A6 expression or localization.

How does S100A6 regulate hematopoietic stem cell self-renewal at the molecular level?

S100A6 regulates HSC self-renewal through multiple interconnected molecular mechanisms:

• S100A6 governs calcium homeostasis, particularly buffering intracellular and mitochondrial calcium levels upon cytokine stimulation

• It activates the Akt pathway, which can be demonstrated by the rescue of S100A6KO phenotypes through Akt activator SC79

• S100A6 affects mitochondrial function and respiratory metabolism, as evidenced by gene set enrichment analysis showing downregulation of mitochondria respiration and electron transport chain pathways in S100A6KO mice

• It interacts with Hsp90 protein complexes, with proteomic analysis revealing downregulation of the HSP90 protein pathway in S100A6KO

• Transcriptomic analysis has shown that genes upregulated in S100A6KO are related to intrinsic apoptotic signaling pathway, explaining the increased apoptosis observed in HSCs

Methodologically, researchers investigating these mechanisms should employ a combination of genetic approaches (conditional knockout models), biochemical assays (protein-protein interactions), and functional readouts (colony formation assays, calcium imaging).

What are the technical considerations when studying S100A6-calcium interactions in murine cells?

When studying S100A6-calcium interactions in murine cells, researchers should consider several technical factors:

• S100A6 functions in a Ca²⁺-dependent manner, requiring careful control of calcium concentrations in experimental buffers

• Direct interaction studies require purified proteins and appropriate calcium concentrations to observe binding events

• For cellular studies, calcium chelators can be used to determine calcium dependency of observed effects

• S100A6 regulates both cytosolic and mitochondrial calcium levels, necessitating compartment-specific calcium measurements

• Different experimental readouts may require different calcium concentration ranges to observe optimal S100A6 activity

• When studying the interaction of S100A6 with other proteins (like cofilin-1), the calcium concentration can significantly affect binding affinity and subsequent functional outcomes

Methodologically, researchers should employ calcium imaging techniques, careful buffer preparation, and appropriate controls to accurately interpret S100A6-calcium dependent phenomena.

How do S100A6 interactions with the cytoskeleton differ between murine cell types?

S100A6 interactions with the cytoskeleton show notable differences across murine cell types:

• In NIH3T3 fibroblasts, S100A6 forms complexes with both actin and tropomyosin, as demonstrated by proximity ligation assays

• S100A6 preferentially interacts with the Tpm1.8 isoform of tropomyosin, suggesting cell-type specific effects based on tropomyosin isoform expression patterns

• The presence of tropomyosin on microfilaments facilitates S100A6 binding, indicating that cell types with different tropomyosin expression profiles may exhibit varying S100A6-cytoskeletal interactions

• In a calcium-bound form, S100A6 can regulate actin filament dynamics by controlling the activity of cofilin-1, which may have differential effects based on cell-type specific cofilin expression and activity

• S100A6 deficiency alters cell morphology, suggesting important cytoskeletal regulatory functions that may manifest differently based on cell type

For accurate comparative studies between cell types, researchers should quantify baseline expression of S100A6, tropomyosin isoforms, and other interaction partners while employing consistent methodological approaches across cell types.

What are the contradictions in current literature regarding S100A6 function in murine models?

Several areas of contradictory findings exist in the S100A6 murine research literature:

• S100A6 and cell survival: While some studies show S100A6 has antiapoptotic effects in certain cell contexts (including murine HSCs) , other research indicates that at micromolar concentrations, S100A6 can trigger cell apoptosis through different signaling pathways than other S100 proteins like S100B

• Receptor interactions: Despite structural similarities with other S100 proteins, S100A6 interacts with different RAGE extracellular domains than S100B, leading to opposing effects on cell survival pathways

• Signaling pathway activation: Some studies emphasize the PI3K/AKT pathway involvement , while others highlight JNK activation , suggesting context-dependent signaling

• Cell-type specific functions: Effects observed in hematopoietic stem cells may differ significantly from those in fibroblasts or other cell types, making cross-comparison difficult

Methodologically, researchers should address these contradictions by clearly defining experimental conditions, concentrations, and cell types when reporting S100A6 functions and avoid overgeneralizing findings across systems.

What experimental approaches can establish causality between S100A6 expression and phenotypic outcomes in murine models?

To establish causality between S100A6 expression and observed phenotypes, researchers should consider:

• Genetic manipulation approaches:

  • Conditional knockout models using systems like Vav-Cre;S100a6 to achieve tissue-specific deletion

  • Rescue experiments using wild-type S100A6 expression in knockout backgrounds

  • Point mutations of critical domains to differentiate calcium-binding functions from other activities

• Pharmacological interventions:

  • Using Akt activators like SC79 to rescue S100A6KO phenotypes, as demonstrated in colony formation assays

  • Calcium modulators to alter S100A6 activity in predictable ways

  • Targeted disruption of specific protein-protein interactions

• Temporal control strategies:

  • Inducible knockout systems to study acute versus chronic loss of S100A6

  • Time-course experiments to establish sequence of molecular events

• Validation across multiple systems:

  • Parallel in vitro and in vivo experiments

  • Cross-validation in multiple cell types

  • Correlation of molecular findings with functional outcomes

For optimal causal inference, researchers should combine multiple approaches and include appropriate controls for each experimental system.

What are the optimal murine models for studying S100A6 function?

Several murine models have proven valuable for S100A6 research:

• Conditional knockout models: The Vav-Cre;S100a6 system has been successfully used to study hematopoietic-specific S100A6 functions

• Cell-specific models: Given S100A6's differential expression across tissues, targeted models for fibroblasts, epithelial cells, or stomach tissues may be particularly informative

• Transgenic overexpression models: Can help identify gain-of-function phenotypes

• Point mutation models: Especially those affecting calcium-binding domains to dissect calcium-dependent versus independent functions

• Reporter models: Combining S100A6 expression with fluorescent reporters can facilitate tracking of expression patterns

When selecting a model, researchers should consider:

• The specific research question and tissues of interest

• Potential developmental effects of S100A6 loss

• Whether acute or chronic manipulation is needed

• The need to distinguish cell-autonomous versus non-cell-autonomous effects

How can researchers accurately quantify changes in calcium homeostasis mediated by S100A6?

To quantify S100A6-mediated calcium homeostasis changes:

• Subcellular calcium measurements:

  • Specific probes for cytosolic versus mitochondrial calcium levels are critical, as S100A6 differentially regulates these compartments

  • Ratiometric calcium indicators provide more reliable quantitative data than single-wavelength indicators

• Real-time versus fixed-point measurements:

  • Live-cell calcium imaging allows tracking of dynamic changes

  • Fixed-point measurements may miss transient calcium fluctuations

• Experimental stimulation:

  • Cytokine stimulation, particularly with SCF (stem cell factor), can reveal S100A6-dependent calcium regulation

  • Controlled calcium influx using ionophores can help establish dose-response relationships

• Analytical considerations:

  • Baseline normalization is essential for comparing across experiments

  • Area-under-curve analysis may better capture total calcium response than peak measurements

  • Statistical approaches should account for the typically non-normal distribution of calcium response data

What methodological approaches best capture S100A6's impact on mitochondrial function?

To effectively measure S100A6's impact on mitochondrial function:

• Respiratory capacity analysis:

  • Oxygen consumption rate (OCR) measurements using platforms like Seahorse XF can quantify changes in mitochondrial respiration

  • Substrate-specific respiration tests can identify which metabolic pathways are most affected

• Mitochondrial membrane potential:

  • Fluorescent probes like TMRM or JC-1 can detect changes in membrane potential that may reflect S100A6-mediated effects

• ROS production:

  • Measuring mitochondrial-specific ROS production can capture oxidative stress effects suggested by transcriptomic analysis of S100A6KO mice

• Mitochondrial calcium:

  • Mitochondrial-targeted calcium indicators can specifically measure the compartmentalized calcium changes regulated by S100A6

• Combined approaches:

  • Correlating multiple mitochondrial parameters with S100A6 expression/activity

  • Rescue experiments with mitochondrial-targeted interventions

When interpreting results, researchers should consider that mitochondrial function varies substantially between cell types and is sensitive to experimental conditions like media composition and cell density.

What are the key considerations when designing experiments to study S100A6 interactions with the Akt pathway?

When studying S100A6-Akt pathway interactions:

• Activation markers:

  • Phosphorylation status of Akt at Ser473 and Thr308 should be quantified

  • Downstream targets of Akt (like FOXO proteins) provide functional readouts

• Temporal dynamics:

  • Early versus late Akt activation may have different dependencies on S100A6

  • Time-course experiments are essential for capturing the full signaling profile

• Pathway specificity:

  • Controls examining parallel pathways help establish specificity

  • Inhibitors of upstream regulators can determine pathway dependency

• Rescue experiments:

  • Akt activators like SC79 have been shown to rescue S100A6KO phenotypes and should be included

  • Dose-response experiments with activators can establish threshold effects

• Functional outcomes:

  • Colony formation assays provide a reliable functional readout for Akt-dependent effects in hematopoietic cells

  • Cell survival/apoptosis assays capture another major Akt-dependent function

Researchers should acknowledge potential cross-talk with other pathways, as S100A6 has multiple interacting partners that could influence Akt signaling indirectly.

How should researchers interpret contradictory data on S100A6 effects in different murine tissues?

When faced with contradictory data on S100A6 effects across tissues:

• Context specificity analysis:

  • Systematically compare experimental conditions (calcium concentrations, cell density, culture conditions)

  • Examine baseline S100A6 expression levels across the tissues being compared

  • Consider the expression of known S100A6 interaction partners (RAGE, tropomyosin isoforms, etc.)

• Dose-response relationships:

  • S100A6 may have different effects at different concentrations, as observed with apoptosis induction at micromolar levels

  • Establish full dose-response curves rather than testing single concentrations

• Pathway analysis:

  • Comparative pathway analysis between tissues may reveal why S100A6 activates different downstream effectors

  • Consider that S100A6 may activate different RAGE domains or receptors in different tissues

• Temporal considerations:

  • Acute versus chronic effects of S100A6 perturbation may differ

  • Developmental timing of S100A6 disruption could impact phenotypic outcomes

Rather than viewing contradictory data as problematic, researchers should use these differences to develop more nuanced models of S100A6 function that account for tissue-specific contexts.

What statistical approaches are most appropriate for analyzing S100A6 expression data in murine samples?

For optimal statistical analysis of S100A6 expression data:

• Data normalization:

  • For transcriptomic data, appropriate housekeeping genes must be selected that aren't affected by experimental conditions

  • For protein quantification, total protein normalization often outperforms single reference protein approaches

• Distribution testing:

  • S100A6 expression often follows non-normal distributions, requiring non-parametric statistical tests

  • Log transformation may be necessary before applying parametric tests

• Multivariable approaches:

  • Principal component analysis can help identify patterns in complex datasets

  • Hierarchical clustering can identify samples with similar S100A6-related expression profiles

• Effect size reporting:

  • Beyond p-values, effect sizes should be reported to assess biological significance

  • Fold changes should be accompanied by confidence intervals

• Multiple testing correction:

  • When analyzing S100A6 alongside other genes, appropriate corrections (like FDR) should be applied

  • Adjusted p-values provide more reliable indicators of significance in large datasets

For comprehensive analysis, researchers should combine multiple statistical approaches and validate findings across independent datasets when possible.

What are the most reliable biomarkers for assessing S100A6 activity in murine experimental systems?

Reliable biomarkers for S100A6 activity include:

• Direct markers:

  • S100A6 protein levels (though these don't necessarily reflect activity)

  • Calcium-bound versus calcium-free S100A6 ratio

  • S100A6 localization (nuclear versus cytoplasmic)

• Functional readouts:

  • Phosphorylated Akt levels, which correlate with S100A6 activity

  • HSP90 pathway activity, which is downstream of S100A6

  • Colony formation capacity in hematopoietic cells

  • Mitochondrial calcium levels

• Transcriptional indicators:

  • Gene sets related to oxidative stress and mitochondrial function

  • Apoptosis-related gene expression

• Phenotypic markers:

  • LT-HSC and MPP population sizes in hematopoietic studies

  • Annexin V/DAPI profiles for apoptosis assessment

  • Cytoskeletal organization patterns

When selecting biomarkers, researchers should choose those most relevant to their specific research question and validate the relationship between the biomarker and S100A6 activity in their experimental system.

How can researchers differentiate between direct and indirect effects of S100A6 in murine experimental models?

To distinguish direct from indirect S100A6 effects:

• Temporal resolution:

  • Time-course experiments can establish the sequence of events following S100A6 perturbation

  • Rapid changes (minutes to hours) are more likely direct effects than changes occurring over days

• Biochemical validation:

  • In vitro binding assays with purified proteins can confirm direct interactions

  • Proximity ligation assays can validate protein-protein interactions in cellular contexts

• Domain mapping:

  • Mutational analysis of interaction domains can confirm specific binding requirements

  • Competition assays with peptides derived from interaction domains can disrupt direct effects

• Rescue experiments:

  • Selective restoration of specific interaction capabilities through domain-swapping experiments

  • Using molecules that bypass S100A6 (like Akt activator SC79) to rescue phenotypes indicates pathway specificity

• Immediate-early responses:

  • Analyzing calcium fluxes, post-translational modifications, or protein relocalization events that occur rapidly after S100A6 manipulation

By combining these approaches, researchers can build a hierarchical model of S100A6 effects, distinguishing primary (direct) from secondary and tertiary (indirect) consequences of S100A6 activity.

How can single-cell technologies advance our understanding of S100A6 function in murine models?

Single-cell technologies offer several advantages for S100A6 research:

• Heterogeneity detection:

  • Single-cell RNA sequencing can reveal subpopulations with differential S100A6 expression that might be missed in bulk analysis

  • Particularly valuable for heterogeneous tissues like bone marrow where S100A6 has important functions

• Multi-parameter correlation:

  • Single-cell proteomics can correlate S100A6 with multiple signaling proteins simultaneously

  • Mass cytometry (CyTOF) can measure S100A6 alongside dozens of other proteins and post-translational modifications

• Spatial context:

  • Spatial transcriptomics can map S100A6 expression patterns within intact tissues

  • Imaging mass cytometry can reveal S100A6 localization relative to interaction partners

• Temporal dynamics:

  • Live-cell imaging of individual cells can track S100A6-dependent processes with high temporal resolution

  • Allows correlation of S100A6 activity with cellular behaviors on a cell-by-cell basis

When implementing these technologies, researchers should develop analysis pipelines specifically optimized for detecting S100A6-related phenomena and validate findings using orthogonal approaches.

What are promising therapeutic targets based on S100A6 pathways in murine disease models?

Based on current understanding of S100A6 functions, promising therapeutic targets include:

• Akt pathway modulators:

  • Akt activators like SC79 have shown promise in rescuing S100A6 deficiency phenotypes

  • Could be therapeutically relevant in conditions with reduced S100A6 function

• Calcium homeostasis regulators:

  • Targeted calcium modulators that mimic S100A6's calcium buffering function

  • Particularly relevant for mitochondrial calcium regulation

• Cytoskeletal dynamics:

  • Compounds affecting the S100A6-tropomyosin-actin interaction might modulate cell migration or morphology

  • Could be relevant in fibrosis models where cell morphology and migration are central

• HSP90 pathway:

  • Given S100A6's interaction with HSP90, HSP90 modulators might compensate for S100A6 dysregulation

  • Could affect protein quality control and stress responses

• Hematopoietic stem cell regulators:

  • Compounds targeting pathways downstream of S100A6 in HSCs could have applications in bone marrow failure or hematologic malignancies

When evaluating potential therapeutic targets, researchers should assess specificity, off-target effects, and the potential consequences of modulating pathways with broad physiological roles.

How can computational modeling enhance experimental approaches to studying S100A6 in murine systems?

Computational modeling can significantly enhance S100A6 research through:

• Structural biology:

  • Molecular dynamics simulations of S100A6-calcium binding

  • Docking studies predicting interactions with partners like actin, tropomyosin, or RAGE

  • Prediction of how mutations affect binding properties

• Systems biology:

  • Network analysis integrating S100A6 into larger signaling networks

  • Prediction of emergent properties from S100A6-regulated pathways

  • Identification of potential feedback mechanisms

• Multi-scale modeling:

  • Linking molecular S100A6 functions to cellular behaviors like migration or differentiation

  • Predicting tissue-level outcomes of S100A6 perturbation

• Data integration:

  • Machine learning approaches to integrate diverse S100A6 datasets

  • Identification of patterns across transcriptomic, proteomic, and functional data

  • Prediction of previously unrecognized S100A6 functions

When developing computational models, researchers should ensure they incorporate known biochemical properties of S100A6 (like calcium dependency) and validate predictions with targeted experiments.

What experimental approaches can resolve the tissue-specific roles of S100A6 in murine development and disease?

To address tissue-specific S100A6 functions:

• Conditional genetic approaches:

  • Tissue-specific Cre lines (beyond the Vav-Cre used for hematopoietic studies)

  • Inducible systems allowing temporal control of S100A6 disruption

  • Intersectional genetics to target specific cell subsets

• Lineage tracing:

  • Tracking the fate of S100A6-expressing cells during development

  • Determining whether S100A6 marks specific progenitor populations

• Parabiosis and transplantation:

  • Distinguishing cell-autonomous versus environmental effects of S100A6

  • Determining whether S100A6-mediated phenotypes are transplantable

• Organoid models:

  • Tissue-specific 3D culture systems to study S100A6 in controlled environments

  • Particularly valuable for tissues with complex cellular composition

• Disease challenge models:

  • Tissue-specific disease induction in S100A6 mutant backgrounds

  • Assessment of how S100A6 perturbation affects disease course in specific tissues

These approaches should be combined with careful phenotyping of multiple tissues to build a comprehensive understanding of S100A6's diverse roles across the organism.

What are the optimal antibodies and detection methods for murine S100A6 in different applications?

Optimal detection strategies for murine S100A6 include:

• Western blotting:

  • Monoclonal antibodies provide highest specificity

  • Important to verify antibodies cross-react with murine S100A6 if raised against human protein

  • Denaturing conditions may affect epitope accessibility

• Immunohistochemistry/Immunofluorescence:

  • Fixation conditions critically impact S100A6 detection (paraformaldehyde preferable to methanol)

  • Antigen retrieval often necessary for formalin-fixed tissues

  • Co-staining with interaction partners provides contextual information

• Flow cytometry:

  • For intracellular S100A6 detection, permeabilization conditions must be optimized

  • Combining with surface markers allows identification of specific cell populations

• Proximity assays:

  • Proximity ligation assay has been successfully used to detect S100A6 complexes with actin and tropomyosin

  • Requires careful antibody validation to avoid false positives

• Mass spectrometry:

  • Label-free quantification or isobaric labeling can provide absolute quantification

  • Consider enrichment strategies for low-abundance S100A6 in complex samples

For all methods, appropriate validation through S100A6KO samples as negative controls is essential to confirm specificity.

What controls and validation steps are essential when conducting S100A6 knockout studies in mice?

Critical controls for S100A6 knockout studies include:

• Genetic validation:

  • Confirmation of deletion at DNA level through PCR genotyping

  • RNA-level validation through qPCR or RNA-seq to confirm absence of transcript

  • Protein-level validation through Western blotting or immunostaining

• Breeding considerations:

  • Littermate controls with matched genetic background are essential

  • Age and sex matching between experimental groups

  • For conditional knockouts, Cre-only controls to account for Cre toxicity

• Phenotypic baselines:

  • Comprehensive phenotyping of knockout mice under steady-state conditions

  • Assessment of developmental effects versus acute phenotypes

• Rescue experiments:

  • Re-expression of S100A6 should rescue phenotypes if directly caused by S100A6 loss

  • Pathway-specific rescue (e.g., Akt activator SC79) validates downstream mechanisms

• Specificity controls:

  • Examination of related S100 family members to rule out compensatory changes

  • Assessment of calcium homeostasis to distinguish direct versus indirect effects

These validation steps ensure that observed phenotypes are specifically attributable to S100A6 loss rather than genetic background effects or technical artifacts.

What are the key technical challenges in studying S100A6-calcium interactions in murine tissues?

Technical challenges in studying S100A6-calcium interactions include:

• Calcium concentration control:

  • Maintaining precise calcium concentrations in experimental buffers

  • Accounting for compartmentalized calcium within cells and organelles

  • Preventing calcium leakage during cell preparation

• Temporal resolution:

  • Calcium signals can be transient, requiring high-speed imaging

  • Correlating calcium dynamics with S100A6 activity in real-time

• Spatial considerations:

  • Distinguishing cytosolic versus mitochondrial calcium pools affected by S100A6

  • Visualizing subcellular localization of calcium-bound versus calcium-free S100A6

• Physiological relevance:

  • Ensuring experimental calcium concentrations reflect physiological ranges

  • Validating in vitro findings in intact tissues

• Technical artifacts:

  • Calcium indicators can buffer calcium themselves, altering the system being measured

  • Tissue processing can disrupt normal calcium distribution

To address these challenges, researchers should combine multiple methodological approaches, carefully control experimental conditions, and validate findings across different detection systems.

How can researchers effectively isolate and study S100A6-expressing populations in murine tissues?

Effective isolation of S100A6-expressing populations requires:

• Flow cytometry strategies:

  • For hematopoietic cells, established markers like LSKCD150⁺CD48⁻ can identify populations with high S100A6 expression

  • Additional markers like CD9 and ESAM have been used for further purification of HSCs

  • Intracellular staining for S100A6 can be combined with surface markers

• Genetic approaches:

  • S100A6 promoter-driven reporter mice expressing fluorescent proteins

  • Cre recombinase driven by the S100A6 promoter for lineage tracing

• Tissue preparation:

  • Optimized digestion protocols for tissues with high S100A6 expression (stomach, fibroblast-rich tissues)

  • Gentle dissociation to maintain cellular integrity

• Enrichment technologies:

  • Magnetic-activated cell sorting (MACS) as a pre-enrichment step

  • c-kit enrichment has been successfully used for HSC populations

• Single-cell approaches:

  • Index sorting to correlate S100A6 expression with other markers

  • Single-cell RNA-seq to identify transcriptional signatures of S100A6-expressing cells

These isolation strategies should be validated by confirming S100A6 expression in the isolated populations through orthogonal methods like qPCR or Western blotting.