LONRF1 Antibody

What is LONRF1 and what cellular functions does it perform?

LONRF1 belongs to the LONRF family of proteins, which consists of three isozymes (LONRF1-3) containing RING domains and a LON substrate-binding domain (LonSB). While LONRF2 has been well-characterized as a protein quality control ubiquitin ligase predominantly acting in neurons, LONRF1 has more diverse expression patterns. LONRF1 appears to play roles in oxidative damage responses and tissue remodeling during wound healing, with potential functions in both senescent and non-senescent cells . Unlike LONRF2, which selectively ubiquitylates misfolded proteins for proteasome-dependent degradation, LONRF1's precise molecular mechanisms remain under investigation.

What is the tissue expression pattern of LONRF1?

LONRF1 is ubiquitously expressed across multiple tissue types. Quantitative PCR analysis using mouse tissue cDNA has revealed that while LONRF1 is present in numerous tissues, its highest expression occurs in the testis . Within the liver, LONRF1 is predominantly expressed in Kupffer cells, liver sinusoidal endothelial cells (LSECs), and hepatocytes. Interestingly, the number of LONRF1 high-expressing cells increases with age in Kupffer cells and LSECs, but not in hepatocytes, suggesting a potential role in age-related processes in non-parenchymal cells .

How does LONRF1 differ from other members of the LONRF family?

LONRF family proteins (LONRF1-3) share structural similarities with their RING domains and LON substrate-binding domains, but they exhibit distinct tissue distribution patterns and functions. LONRF2 is predominantly expressed in the brain and functions as a protein quality control ubiquitin ligase, with LONRF2-deficient mice exhibiting late-onset neurological deficits . In contrast, LONRF1 is widely expressed across tissues and appears to be involved in antioxidant responses and tissue remodeling. Human LONRF2 contains two RING domains, whereas mouse LONRF2 contains only one, demonstrating species-specific differences even within the same family member .

What criteria should be considered when selecting a LONRF1 antibody for research?

When selecting a LONRF1 antibody, researchers should evaluate several key parameters:

• Target species compatibility (e.g., human, mouse, rat)

• Application validation (IHC, WB, ICC/IF, ELISA)

• Clonality (monoclonal vs. polyclonal)

• Epitope location and accessibility

• Validation data quality

• Cross-reactivity profile with other LONRF family members

• Publication record in peer-reviewed research

The antibody should be validated for your specific application and experimental conditions. For example, some commercially available LONRF1 antibodies have been validated for immunohistochemistry and western blotting in human samples .

How can I validate a LONRF1 antibody for my specific experimental conditions?

Antibody validation should include multiple approaches:

• Positive control testing: Use tissues or cell lines known to express LONRF1 (testis tissue shows high expression levels)

• Negative control testing: Use tissues with low/no LONRF1 expression or LONRF1 knockout models

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of the pulled-down protein

• siRNA knockdown: Compare signal between LONRF1-silenced and control samples

• Cross-validation: Use multiple antibodies targeting different epitopes

• Orthogonal methods: Confirm protein expression with mRNA expression data

For LONRF1, which is ubiquitously expressed but highest in testis tissue, comparing signal intensities across multiple tissues can provide additional validation evidence .

What are the recommended protocols for detecting LONRF1 by Western blotting?

For optimal Western blot detection of LONRF1:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if investigating post-translational modifications

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels (LONRF1 molecular weight: ~80 kDa)

  • Load 20-50 μg of total protein per lane

  • Include positive control (testis tissue extract)

• Transfer and blocking:

  • Transfer to PVDF membrane at 100V for 90 minutes

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary: Anti-LONRF1 antibody (1:1000 dilution) overnight at 4°C

  • Secondary: HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) substrate

  • Expected band size: approximately 80 kDa

Include β-actin or GAPDH as loading controls, and verify results using tissues with known differential expression patterns of LONRF1 (e.g., testis vs. other tissues) .

How can I optimize LONRF1 immunohistochemistry staining protocols?

For optimized IHC staining of LONRF1:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow to cool to room temperature before proceeding

• Blocking and permeabilization:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Permeabilize with 0.2% Triton X-100 in PBS for 15 minutes

  • Block non-specific binding with 5% normal serum for 1 hour

• Antibody incubation:

  • Primary: Anti-LONRF1 antibody diluted 1:100-1:500 in blocking buffer, overnight at 4°C

  • Secondary: HRP-conjugated secondary antibody for 1 hour at room temperature

• Detection and visualization:

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Mount and observe

Include positive controls (testis tissue) and negative controls (primary antibody omission) . When analyzing liver tissue, pay particular attention to Kupffer cells and LSECs, which show age-dependent increases in LONRF1 expression .

What is the recommended approach for using LONRF1 antibodies in immunofluorescence?

For immunofluorescence detection of LONRF1:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash thoroughly with PBS

• Permeabilization and blocking:

  • Permeabilize with 0.2% Triton X-100 in PBS for 15 minutes

  • Block with 5% normal serum in PBS for 1 hour at room temperature

• Antibody incubation:

  • Primary: Anti-LONRF1 antibody (1:100-1:200) overnight at 4°C

  • Secondary: Fluorophore-conjugated secondary antibody (1:500) for 1 hour at room temperature, protected from light

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

• Co-localization studies:

  • For liver tissue, co-stain with markers for Kupffer cells (F4/80) or LSECs (CD31)

  • For fibroblasts, consider co-staining with p16 to distinguish between Lonrf1 high/p16 high and Lonrf1 high/p16 low populations

When analyzing LONRF1 subcellular localization, note that LONRF1 does not appear to contain a nuclear localization signal similar to that found in Lonp1 , so expect predominantly cytoplasmic localization.

How can LONRF1 antibodies be used to study age-related changes in liver function?

LONRF1 expression increases with age in liver sinusoidal endothelial cells (LSECs) and Kupffer cells, making it a valuable marker for studying age-related changes in liver function . A comprehensive approach would include:

• Age-comparative analysis:

  • Collect liver samples from young, middle-aged, and old animals

  • Perform immunohistochemistry using anti-LONRF1 antibodies

  • Quantify LONRF1-positive cells as a percentage of total cells by cell type

• Cell type-specific isolation:

  • Isolate Kupffer cells and LSECs using magnetic bead separation

  • Compare LONRF1 protein levels by Western blotting

  • Correlate with functional assays of phagocytic capacity and antioxidant response

• Single-cell analysis workflow:

  • Perform single-cell RNA sequencing of liver tissue

  • Identify LONRF1 high and LONRF1 low populations

  • Compare transcriptional profiles between age groups

• Functional correlation:

  • Assess peptidase activity in Kupffer cells, which is enriched in LONRF1 high cells

  • Evaluate NF-κB and p53 pathway activation in LSECs

  • Measure oxidative stress markers and correlate with LONRF1 expression levels

This approach can reveal how LONRF1 expression changes contribute to age-related alterations in liver function, particularly in non-parenchymal cells that play critical roles in liver homeostasis .

What is the relationship between LONRF1 expression and oxidative stress responses?

LONRF1 appears to play a protective role in oxidative stress responses. Based on transcriptomic analysis, LONRF1 high cells show suppression of cytoprotection by HMOX1, a primary antioxidant enzyme regulated by the KEAP1-NRF2 system under oxidative stress . A comprehensive investigation would include:

• Oxidative stress induction:

  • Treat cells with H₂O₂, tert-butyl hydroperoxide, or paraquat

  • Measure LONRF1 protein levels by Western blotting

  • Correlate with markers of oxidative damage (8-OHdG, protein carbonylation)

• Pathway analysis:

  • Evaluate activation status of NF-κB and p53 pathways in LONRF1 high vs. low cells

  • Assess IFNα and IFNγ signaling suppression in LONRF1 high cells

  • Examine proteasome activity in relation to LONRF1 expression

• LONRF1 manipulation:

  • Overexpress or knock down LONRF1 in relevant cell types

  • Challenge with oxidative stressors

  • Measure cell viability, ROS production, and antioxidant enzyme activities

• Comparative analysis with LONRF2:

  • Examine expression patterns during oxidative stress

  • Determine functional redundancy or specificity

  • Assess substrate specificity differences

Understanding the relationship between LONRF1 and oxidative stress could provide insights into protective mechanisms against age-related oxidative damage, particularly in liver and fibroblast populations .

How does LONRF1 contribute to tissue remodeling during wound healing?

LONRF1 appears to play distinct roles in different fibroblast populations during wound healing. In p16-low fibroblasts, high LONRF1 expression is associated with cell growth activation and suppression of TGFβ and BMP signaling, while in p16-high fibroblasts, high LONRF1 expression correlates with WNT signaling activation . To study this process:

• Wound healing model:

  • Create standardized wounds in animal models

  • Collect tissue at various time points (days 3, 7, 14)

  • Perform immunohistochemistry for LONRF1 and p16

  • Co-stain with markers of proliferation and differentiation

• Fibroblast subpopulation analysis:

  • Isolate fibroblasts from wound tissue

  • Sort into LONRF1 high/p16 low and LONRF1 high/p16 high populations

  • Compare gene expression profiles by RNA-seq

  • Validate key pathway differences by qPCR and Western blotting

• Functional assays:

  • Assess proliferation rates in each population

  • Measure collagen production and contractility

  • Evaluate myofibroblast differentiation capacity

  • Test response to WNT and TGFβ pathway modulators

• In vitro wound model:

  • Perform scratch assays with LONRF1-manipulated fibroblasts

  • Track wound closure rates and dynamics

  • Correlate with LONRF1 expression levels

This comprehensive approach can elucidate how LONRF1 contributes to tissue remodeling through differential effects on distinct fibroblast populations during wound healing .

What are common issues when detecting LONRF1 by Western blot and how can they be resolved?

For optimal results, use fresh tissue samples and include age-matched controls when studying age-dependent expression changes in liver cells .

How can I distinguish between LONRF1 and other LONRF family members in my experiments?

Distinguishing between LONRF family members requires careful experimental design:

• Antibody selection:

  • Choose antibodies targeting unique epitopes not conserved between LONRF1, LONRF2, and LONRF3

  • Validate specificity using overexpression systems of each family member

• Expression pattern comparison:

  • LONRF2 is predominantly expressed in brain tissue, while LONRF1 is ubiquitous with highest expression in testis

  • Use tissue-specific expression patterns to validate antibody specificity

• Molecular weight verification:

  • LONRF family members have slightly different molecular weights

  • Use high-resolution SDS-PAGE to distinguish between them

• Knockout/knockdown validation:

  • Generate specific knockdowns of each family member

  • Verify antibody specificity by confirming signal loss only in the appropriate knockdown

• Peptide competition assay:

  • Pre-incubate antibody with peptides specific to each family member

  • Only the specific peptide should abolish genuine signal

This approach ensures accurate identification of LONRF1 without cross-reactivity with other family members, which is essential for functional studies .

What controls should be included when studying LONRF1 in different age groups or disease models?

When studying LONRF1 across age groups or disease models, include these essential controls:

• Positive and negative tissue controls:

  • Positive: Testis tissue (high LONRF1 expression)

  • Negative: Tissues with minimal LONRF1 expression or LONRF1 knockout samples

• Age-matched controls:

  • For studies of age-dependent changes in liver cells

  • Include young, middle-aged, and old samples for comprehensive analysis

• Technical controls:

  • Antibody specificity: Primary antibody omission

  • Background assessment: Secondary antibody only

  • Loading controls: Housekeeping proteins for Western blots

• Disease model validation:

  • For NASH studies: Confirm disease phenotype by histopathology

  • For wound healing: Include unwounded tissue as baseline

• Cell type-specific markers:

  • When studying liver: Include markers for Kupffer cells and LSECs

  • When studying wound healing: Include markers for fibroblast subpopulations and p16

• Pathway activation controls:

  • For oxidative stress studies: Include positive controls with known oxidative stress inducers

  • For NF-κB and p53 pathway analyses: Include samples with known pathway activators

These controls ensure reliable interpretation of results when studying LONRF1's role in aging and disease processes .

What are promising approaches to study LONRF1's role in protein quality control?

Given that LONRF2 functions as a protein quality control ubiquitin ligase and LONRF1 shares structural domains, investigating LONRF1's potential role in protein quality control is warranted:

• Substrate identification:

  • Perform immunoprecipitation of LONRF1 followed by mass spectrometry

  • Conduct BioID or proximity labeling to identify interacting proteins

  • Compare substrates with those of LONRF2 to identify unique targets

• Ubiquitination assays:

  • Develop in vitro ubiquitination assays with recombinant LONRF1

  • Identify E2 conjugating enzymes that cooperate with LONRF1

  • Determine ubiquitin chain linkage specificity

• Proteostasis challenge models:

  • Subject cells to proteotoxic stress (heat shock, proteasome inhibitors)

  • Compare responses in LONRF1-depleted vs. control cells

  • Assess protein aggregation and clearance kinetics

• Domain function analysis:

  • Generate domain mutants (RING domain, LonSB domain)

  • Assess the contribution of each domain to LONRF1 function

  • Compare with equivalent domains in LONRF2 and LONRF3

These approaches would provide insights into whether LONRF1 functions similarly to LONRF2 in protein quality control or has evolved distinct functions .

How might LONRF1 be involved in age-related pathologies beyond liver disease?

Given LONRF1's ubiquitous expression and age-dependent regulation in certain cell types, it may play roles in various age-related pathologies:

• Neurodegenerative diseases:

  • Examine LONRF1 expression in brain tissues from Alzheimer's and Parkinson's disease models

  • Compare with LONRF2, which is known to be neuroprotective

  • Investigate potential compensation between family members

• Cardiovascular aging:

  • Study LONRF1 expression in vascular endothelial cells during aging

  • Analyze correlation with markers of endothelial dysfunction

  • Investigate role in vascular remodeling and atherosclerosis

• Immune system senescence:

  • Examine LONRF1 expression in aging immune cells

  • Correlate with inflammatory phenotypes

  • Investigate role in inflammaging processes

• Cancer biology:

  • Analyze LONRF1 expression in tumor vs. normal tissues

  • Investigate relationship with cellular senescence in tumor microenvironment

  • Explore potential roles in therapy resistance

• Fibrotic disorders:

  • Based on LONRF1's role in fibroblasts during wound healing

  • Study expression in fibrotic tissues across multiple organs

  • Investigate therapeutic potential of LONRF1 modulation

These investigations could reveal new roles for LONRF1 in age-related pathologies and potentially identify novel therapeutic targets .