erlin2 Antibody

What is ERLIN2 and why is it significant in cancer research?

ERLIN2 is an endoplasmic reticulum protein that regulates several critical cellular processes. It has garnered significant research interest because the ERLIN2 gene is amplified and overexpressed in aggressive human breast cancers . The protein supports cancer cell growth by regulating cytosolic lipid droplet production and facilitating the transformation of human breast cancer cells . Additionally, ERLIN2 functions as an ER-microtubule-binding protein that stabilizes mitosis-promoting factors, particularly through its interaction with the Cyclin B1/Cdk1 complex in G2/M phase . Unlike many proteins, ERLIN2 shows developmental regulation—with high expression postnatally that becomes nearly undetectable in adult tissues . This expression pattern, combined with its role in lipid metabolism and cell cycle regulation, makes ERLIN2 a compelling target for understanding cancer cell metabolism and proliferation.

What are the optimal sample preparation methods for ERLIN2 antibody applications?

For effective ERLIN2 antibody applications, sample preparation should account for ERLIN2's subcellular localization and interactions. When preparing protein extracts for Western blotting, NP-40 lysis has proven effective for total cell lysates . For immunoprecipitation studies, protein lysates should be prepared under conditions that preserve protein-protein interactions.

When studying ERLIN2's interaction with microtubules, temperature consideration is crucial:

• For detection of ERLIN2-tubulin interactions: Use ice-cold protein lysates to maintain depolymerized microtubules into α- and β-tubulins

• For microtubule polymerization studies: Process samples at 37°C to induce microtubule polymerization

For immunofluorescence studies of ERLIN2's association with microtubules, fixation methods must preserve both ER structure and microtubule integrity, with acetylated-α-tubulin serving as an effective marker for polymerized microtubules .

What validation steps are essential to ensure ERLIN2 antibody specificity?

To ensure reliable research results with ERLIN2 antibodies, comprehensive validation is critical:

• Knockout/knockdown validation: Compare antibody reactivity in ERLIN2-expressing vs. ERLIN2-knockdown cells (as demonstrated in SUM225 cell lines)

• Peptide competition assays: For antibodies generated against synthetic peptides (like ABIN1537101, which targets AA 307-333)

• Cross-reactivity assessment: Test reactivity against the homologous protein ERLIN1, as these proteins interact to form functional complexes

• Multiple antibody comparison: Use antibodies targeting different epitopes of ERLIN2 to confirm results

• Species reactivity verification: Confirm species-specific reactivity as documented in product information

How can researchers effectively study ERLIN2's role in lipid metabolism using antibody-based approaches?

ERLIN2's function in lipid metabolism can be studied through multiple antibody-based approaches:

• Temporal expression analysis: Compare ERLIN2 protein levels across normal and lipid-challenged conditions using Western blotting. Research shows ERLIN2 is inducible by insulin signaling or when cells are cultured in lipoprotein-deficient medium (LPDS) .

• Subcellular fractionation with co-localization: Use ERLIN2 antibodies in conjunction with markers for lipid droplets and ER to track ERLIN2's localization during lipid droplet formation.

• Co-immunoprecipitation studies: Investigate ERLIN2's interaction with SREBP (sterol regulatory element-binding protein) 1c pathway components. ERLIN2 has been shown to regulate activation of SREBP1c, a key regulator of de novo lipogenesis .

• Comparative studies across tissue types: ERLIN2 levels are higher in steatotic livers from mice fed atherogenic high-fat diets compared to those fed normal chow . Researchers should include appropriate tissue controls when designing experiments:

  • Positive controls: Postnatal tissues, aggressive breast cancer cell lines (SUM225, ZR-75-1), hepatoma cell lines (HepG2, Huh-7)

  • Negative/low expression controls: Adult normal tissues, non-transformed mammary epithelial cells (MCF10A), murine primary hepatocytes

• Knockdown-rescue experiments: Combine ERLIN2 antibody detection with functional assays after knockdown and subsequent rescue with wild-type or mutant ERLIN2 to identify essential domains for lipid regulation.

What are the methodological approaches for investigating ERLIN2's interaction with microtubules?

To effectively study ERLIN2's interaction with microtubules, researchers should consider these methodological approaches:

• Co-immunoprecipitation under specific temperature conditions:

  • IP-Western blot analysis with ice-cold protein lysates effectively detects the interaction between ERLIN2 and α-tubulin

  • ERLIN2 interacts specifically with α-tubulin but not β-tubulin in breast cancer cells

• Immunofluorescence co-localization:

  • Use antibodies against ERLIN2 and acetylated-α-tubulin (marker for polymerized microtubules)

  • Confocal microscopy reveals significant colocalization of ERLIN2 with α-tubulin in microtubules

• Microtubule stability assays:

  • Challenge cells with microtubule-depolymerizing agents (e.g., nocodazole)

  • Compare microtubule integrity in ERLIN2-knockdown vs. control cells

  • Research shows that microtubules are more susceptible to disruption in ERLIN2-knockdown cells

• Microtubule cosedimentation assays:

  • Incubate ERLIN2-enriched membrane protein fractions with microtubule extracts

  • Test under various conditions: 0°C (depolymerization) vs. 37°C (polymerization)

  • Presence of ERLIN2 in pellets with polymerized microtubules confirms association

  • Include controls: cytoplasmic proteins (IRS1), other ER membrane proteins (SCAP, TRC8, GP78)

• Recovery assays:

  • Treat cells with nocodazole, then release to allow microtubule repolymerization

  • Monitor acetylated α-tubulin levels during recovery as an indicator of microtubule stability

  • ERLIN2-knockdown cells show lower acetylated α-tubulin levels after release from nocodazole

How can researchers address the discrepancy between ERLIN2 protein and mRNA expression levels?

Studies have identified a notable discrepancy between ERLIN2 protein and mRNA expression levels in certain contexts . To investigate this phenomenon:

• Parallel quantification protocols:

  • Perform quantitative real-time RT-PCR for mRNA quantification

  • Utilize Western blot with proper loading controls for protein quantification

  • Compare expression ratios across different tissues and conditions

• Post-transcriptional regulation analysis:

  • Investigate microRNA binding to ERLIN2 mRNA using prediction tools and validation assays

  • Assess mRNA stability through actinomycin D chase experiments

  • Examine translational efficiency using polysome profiling

• Protein stability assessment:

  • Conduct cycloheximide chase experiments to determine ERLIN2 protein half-life

  • Investigate proteasomal and lysosomal degradation pathways

  • Compare degradation rates across cell types where discrepancies are observed

• Experimental controls:

  • Include genes with known post-transcriptional regulation in lipid metabolism pathways as positive controls, as this discrepancy has been observed with many genes involved in lipid metabolism

  • Validate findings using multiple cell lines and tissue samples

What methodologies are most effective for studying ERLIN2's role in cell cycle regulation?

ERLIN2 plays a critical role in cell cycle progression, particularly through interactions with the mitosis-promoting complex Cyclin B1/Cdk1 . To investigate this function:

• Cell cycle synchronization and analysis:

  • Synchronize cells at G2/M phase using nocodazole or thymidine blocks

  • Use flow cytometry to confirm cell cycle phase distribution

  • Analyze ERLIN2 protein levels and interactions at different cell cycle phases

• Co-immunoprecipitation studies:

  • Pull down ERLIN2 to detect associated Cyclin B1/Cdk1 complex

  • Research shows this interaction is maximal during G2/M phase

  • Include appropriate controls (IgG, input lysate)

• Ubiquitination assays:

  • ERLIN2 facilitates K63-linked ubiquitination and stabilization of Cyclin B1 protein in G2/M phase

  • Use ubiquitination-specific antibodies in combination with ERLIN2 and Cyclin B1 antibodies

  • Compare ubiquitination patterns in ERLIN2-knockdown vs. control cells

• Cell proliferation and malignancy assessment:

  • Monitor G2/M phase progression in ERLIN2-knockdown cells

  • Downregulation of ERLIN2 leads to G2/M phase arrest and represses human breast cancer cell proliferation

• Domain mapping experiments:

  • Create ERLIN2 truncation constructs to identify regions required for interaction with cell cycle proteins

  • Use co-immunoprecipitation and functional assays to validate findings

How can ERLIN2 antibodies be utilized in breast cancer research?

ERLIN2 is significantly relevant to breast cancer research, as it is amplified and overexpressed in aggressive forms . Researchers can utilize ERLIN2 antibodies in several cancer research applications:

• Tissue microarray analysis:

  • Use ERLIN2 antibodies for immunohistochemistry on breast cancer tissue microarrays

  • Correlate expression with clinical parameters (tumor grade, size, metastasis)

  • Statistical analysis has identified ERLIN2 as one of several candidate oncogenes within the 8p11-12 amplicon

• Functional studies in breast cancer models:

  • Compare ERLIN2 expression in aggressive human breast cancer cell lines (SUM225, ZR-75-1, SUM44, SUM52) versus non-transformed mammary epithelial cells (MCF10A)

  • Establish ERLIN2 knockdown in breast cancer cell lines for proliferation and transformation assays

  • Monitor changes in lipid metabolism and cell cycle progression

• Mechanistic investigations:

  • Study ERLIN2's regulation of SREBP1c activation in breast cancer cells

  • Examine ERLIN2's role in stabilizing Cyclin B1, which is associated with high breast tumor grade, larger tumor size, and higher metastasis probability

  • Investigate ERLIN2's interaction with ER-resident proteins (GP78) and other ERAD (ER-associated degradation) components

• Therapeutic target assessment:

  • Use ERLIN2 antibodies to monitor protein levels following experimental therapies

  • Evaluate potential for ERLIN2 as a biomarker for aggressive breast cancer

What are the recommended protocols for using ERLIN2 antibodies in various applications?

Based on the research data and antibody specifications, here are optimized protocols for different applications:

Western Blotting Protocol for ERLIN2 Detection:

• Sample preparation: Use NP-40 lysis buffer

• Protein separation: 10% Tris-Glycine polyacrylamide gels are suitable

• Transfer: 0.45-mm PVDF membrane is recommended

• Detection: Enhanced chemiluminescence detection reagents

• Loading controls: α-tubulin and GAPDH have been successfully used

Immunoprecipitation Protocol:

• Prepare total protein lysates from cultured cells

• Immunoprecipitate with anti-ERLIN2 antibody

• Perform Western blot analysis with antibodies against interaction partners (e.g., α-tubulin, Cyclin B1)

• For detecting interactions with α-tubulin, maintain ice-cold conditions during lysate preparation

Immunofluorescence Protocol:

• Fixation: Methods must preserve both ER structure and microtubule integrity

• Co-staining: ERLIN2 with acetylated-α-tubulin for microtubule studies

• Visualization: Confocal microscopy for co-localization analysis

How can researchers distinguish between ERLIN1 and ERLIN2 functions in experimental settings?

ERLIN1 and ERLIN2 interact to form a functional complex , making it challenging to distinguish their individual functions. To address this:

• Selective antibody targeting:

  • Use antibodies that specifically target unique regions of ERLIN2 (such as AA 307-333)

  • Validate antibody specificity against both ERLIN1 and ERLIN2

• Knockdown specificity:

  • Use siRNA or shRNA specific to either ERLIN1 or ERLIN2

  • Confirm knockdown specificity by Western blot for both proteins

  • The Expression Arrest GIPZ lentiviral shRNAmir system has been successfully used for ERLIN2 knockdown

• Rescue experiments:

  • After knockdown of either protein, perform rescue with wild-type or mutant versions

  • Analyze if ERLIN1 can compensate for ERLIN2 loss and vice versa

• Interaction analysis:

  • Use IP-Western blot to examine ERLIN1-ERLIN2 complex formation under different conditions

  • Map interaction domains to identify unique binding partners for each protein

• Developmental expression patterns:

  • Compare ERLIN1 and ERLIN2 expression across development and in disease states

  • Look for tissues or conditions where one protein is expressed without the other

How can researchers approach studying the developmental regulation of ERLIN2?

ERLIN2 demonstrates strong developmental regulation, being highly expressed at postnatal stages but becoming undetectable in adulthood in normal tissues . Researchers can investigate this phenomenon through:

• Temporal expression profiling:

  • Analyze ERLIN2 expression in multiple tissues at different developmental stages

  • Include embryonic (E14), postnatal (P1, P7, P15), and adult samples

  • Use both Western blot for protein and qRT-PCR for mRNA analysis

• Epigenetic regulation studies:

  • Investigate DNA methylation patterns at ERLIN2 promoter regions

  • Examine histone modifications associated with ERLIN2 gene silencing during development

  • Compare to other developmentally regulated genes

• Transcriptional regulation analysis:

  • Identify transcription factors that bind to ERLIN2 promoter at different developmental stages

  • Perform chromatin immunoprecipitation (ChIP) experiments

  • Investigate the role of developmental signaling pathways in ERLIN2 regulation

• Tissue-specific expression patterns:

  • Research shows ERLIN2 is highly expressed in cerebrum, cerebellum, spinal cord, lung, liver, spleen, and kidney at postnatal day 1

  • Create expression atlases across tissues and developmental timepoints

• Reactivation studies:

  • Investigate conditions that reactivate ERLIN2 expression in adult tissues (e.g., high-fat diet induces ERLIN2 expression in adult mouse liver)

  • Explore pathological conditions that trigger ERLIN2 reexpression

What approaches can address the challenge of studying ERLIN2's dual roles in lipid metabolism and cell cycle regulation?

ERLIN2 has dual functions in regulating lipid metabolism and cell cycle progression. To effectively study these interconnected roles:

• Temporal segregation experiments:

  • Synchronize cells at different cell cycle phases and analyze ERLIN2's lipid regulatory function

  • Determine if ERLIN2's lipid regulatory role changes throughout the cell cycle

• Domain-specific analysis:

  • Generate domain-specific mutants of ERLIN2

  • Identify which domains are responsible for lipid regulation versus cell cycle functions

  • Test mutants in rescue experiments after ERLIN2 knockdown

• Interaction network mapping:

  • Use proximity labeling techniques (BioID, APEX) with ERLIN2 as bait

  • Identify different interactome networks during various cellular processes

  • Compare interaction partners during lipid stress versus mitosis

• Multi-omics integration:

  • Combine lipidomics, proteomics, and transcriptomics data from ERLIN2-manipulated cells

  • Build pathway models that integrate lipid metabolism with cell cycle progression

  • Identify metabolic changes that correlate with cell cycle alterations

• Disease model studies:

  • Compare ERLIN2 function in cancer cells (where both functions may be hyperactivated)

  • Investigate how these dual roles contribute to cancer cell survival and proliferation

  • Develop dual-targeting strategies for potential therapeutic interventions