ERLIN1 Antibody

What is ERLIN1 and where is it typically expressed?

ERLIN1 is a ~40-kDa type II membrane protein located in detergent-resistant membranes (DRMs) within the endoplasmic reticulum. It functions primarily as part of the erlin1/2 complex, a ~2-MDa ensemble formed with ERLIN2. This complex is best characterized for its role in mediating the ubiquitination of activated IP3 receptors .

ERLIN1 protein expression has been detected in multiple tissues and cell lines including:

• Human tissues: kidney, brain, ovary cancer cells, placenta

• Mouse tissues: liver, heart

• Rat tissues: pancreas

• Cell lines: HL-60, COLO 320, U-251, U-397, HeLa

The observed molecular weight of ERLIN1 is approximately 39-41 kDa, depending on the detection system used .

What are the recommended applications and dilutions for ERLIN1 antibodies?

Based on commercial antibody information and research publications, ERLIN1 antibodies can be used for multiple applications with the following recommended dilutions:

For optimal results, it is advised to titrate the antibody concentration for each specific application and sample type .

How can I optimize Western blot protocols for ERLIN1 detection?

For successful Western blot detection of ERLIN1:

• Sample preparation:

  • Use NP-40 lysis buffer for total cell lysates as described in multiple studies

  • For membrane-specific analysis, consider isolating membrane fractions to enrich for ERLIN1

• Electrophoresis:

  • Use 10% Tris-Glycine polyacrylamide gels for optimal separation

  • Transfer proteins to a 0.45-mm PVDF membrane for better retention of target proteins

• Antibody incubation:

  • Start with 1:1000 dilution for most commercial ERLIN1 antibodies

  • For Cell Signaling Technology antibody #2958, the recommended dilution is 1:1000

• Detection considerations:

  • Enhanced chemiluminescence detection reagents provide good sensitivity for ERLIN1

  • Be aware that some pan-antibodies against erlin1/2 may cross-react with unrelated proteins, especially in total cell lysates. This issue can be mitigated by analyzing membrane preparations

What positive controls should I use when working with ERLIN1 antibodies?

When validating ERLIN1 antibodies, the following positive controls have been experimentally verified:

For Western blot:

• Cell lines: HL-60 cells, COLO 320 cells, U-251 cells, U-397 cells, HeLa cells

• Tissues: human kidney tissue, human brain tissue, mouse liver tissue, rat pancreas tissue

For immunoprecipitation:

• Mouse heart tissue has shown reliable results

For IHC applications:

• Mouse liver tissue and human placenta tissue are recommended

A critical validation approach is to use ERLIN1 knockout (KO) samples as negative controls. Several studies have generated ERLIN1 KO cell lines using CRISPR/Cas9 techniques. Single-cell sorting of transfected cells followed by clonal expansion and PCR validation can confirm successful ERLIN1 gene editing .

How can I troubleshoot non-specific bands when detecting ERLIN1?

Non-specific bands are a common challenge when working with ERLIN1 antibodies. Consider these troubleshooting approaches:

• Cross-reactivity issues:

  • Some pan-antibodies that recognize both ERLIN1 and ERLIN2 strongly cross-react with unrelated proteins, particularly in total cell lysates

  • Solution: Use membrane preparations instead of total cell lysates to reduce this cross-reactivity

• Antibody specificity:

  • Validate antibody specificity using ERLIN1 knockout or knockdown samples

  • When using siRNA knockdown for validation, note that multiple siRNA sequences targeting ERLIN1 have been validated, including siErlin 1.5: CCACAAATAGGAGCAGCAT

• Detection of multiple isoforms:

  • Be aware that ERLIN1 (41 kDa) migrates slightly differently than ERLIN2 (43 kDa) on SDS-PAGE

  • The ratio of ERLIN2 to ERLIN1 in αT3 cell membrane extracts is approximately 2:1

• Sample preparation optimization:

  • For difficult samples, consider using detergent-resistant membrane (DRM) isolation by flotation using an Optiprep gradient, which can help concentrate ERLIN1/2 in specific fractions

What are effective strategies for ERLIN1 knockdown studies?

For siRNA-mediated ERLIN1 knockdown:

• Validated siRNA sequences:

  • siErlin 1.5: CCACAAATAGGAGCAGCAT

  • For simultaneous knockdown of ERLIN1 and ERLIN2: siErlin 1&2: AGAAGCAATGGCCTGGTAC

• Transfection protocol:

  • In published studies, 3 × 10^4 cells were plated on 48-well plates

  • 20 hours later, cells were transfected with siRNAs

  • Knockdown efficiency evaluation at 36 hours post-transfection showed significant reduction in protein levels

• Validation of knockdown:

  • Western blotting is the preferred method to confirm knockdown efficiency

  • Important controls include a non-targeting siRNA (example: siCtrol: ACTGTCACAAGTACCTACA)

For CRISPR/Cas9-mediated ERLIN1 knockout:

• Single-cell sorting of transfected cells is recommended, with culture in 50% conditioned media and 10-μM ROCK inhibitor

• Clonal expansion (approximately 3 weeks) and validation by PCR targeting the edited region (e.g., exon 6)

• Verification of editing via TIDE analysis to confirm mutations resulting in early stop codons

How does ERLIN1 interact with ERLIN2 in complex formation?

The erlin1/2 complex formation is essential for its biological function:

• Complex composition:

  • ERLIN1 and ERLIN2 form a ~2-MDa complex in the ER membrane

  • The ratio of ERLIN2 to ERLIN1 in the complex is approximately 2:1, as determined by both immunoprecipitation studies and mass spectral analysis

  • This ratio appears to be maintained in complexes associated with activated IP3R1

• Functional significance:

  • While ERLIN2 alone can bind to activated IP3R1 and recruit RNF170, this complex functions inefficiently compared to the complete erlin1/2 complex

  • ERLIN1 alone cannot bind to activated IP3R1, indicating that ERLIN2 is essential for IP3R1 recognition

  • The T65I mutation in ERLIN2 prevents the formation of a functional erlin1/2 complex with RNF170

• Experimental approaches to study the complex:

  • Immunoprecipitation with either anti-ERLIN1 or anti-ERLIN2 antibodies can pull down the entire complex

  • Recovery of detergent-resistant membranes (DRMs) after solubilization with non-ionic detergent (Triton-X100) by flotation using an Optiprep gradient can isolate the native complex

What is the role of ERLIN1 in IP3R regulation and ERAD pathways?

ERLIN1 plays a crucial role in regulating IP3 receptors through the ERAD pathway:

• IP3R ERAD mechanism:

  • The erlin1/2 complex mediates the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IP3Rs)

  • Upon activation (e.g., by GnRH in αT3 cells), the erlin1/2 complex-RNF170 module associates with IP3Rs

  • This association leads to IP3R ubiquitination and subsequent degradation by proteasomes

• Experimental evidence:

  • In ERLIN1 knockout (E1KO) cells, IP3R1 ubiquitination and down-regulation are substantially inhibited (by ~67%)

  • In ERLIN2 knockout (E2KO) cells, IP3R1 ubiquitination is almost completely blocked (by ~94%)

  • Interestingly, IP3R1 levels are increased by ~73% and ~94% in E1KO and E2KO cells, respectively, suggesting erlins also play a role in basal IP3R1 turnover

• Molecular interactions:

  • ERLIN1/2 scaffolds bridge TMUB1 and RNF170, which are critical for the ERAD process

  • ERLIN scaffolds are specifically required for the interaction between TMUB1-L (long isoform) and RNF170

  • TMUB1-L contains a UBL domain and acts as an escortase stabilizing intermediates of membrane proteins during retro-translocation

What is the connection between ERLIN1 and viral infections like HCV?

ERLIN1 has been identified as a host factor required for efficient hepatitis C virus (HCV) infection:

• Impact on HCV lifecycle:

  • Silencing of ERLIN1 expression by siRNA leads to decreased HCV infection efficiency

  • This is characterized by reduction in:

    • Intracellular HCV RNA accumulation

    • HCV protein expression

    • Infectious virus production

• Mechanism of action:

  • ERLIN1 is required early in the infection, downstream of cell entry and primary translation

  • It specifically facilitates the initiation of HCV RNA replication

  • ERLIN1 also supports infectious virus production at later stages of infection

• Experimental approaches to study ERLIN1 in viral infections:

  • siRNA-mediated knockdown of ERLIN1 in permissive cell lines

  • Analysis of viral RNA levels by RT-qPCR

  • Measurement of viral protein expression by Western blotting

  • Quantification of infectious virus production using reporter systems

  • Controls with pseudotyped retroviral particles (HCVpp or VSVpp) to distinguish between effects on entry versus post-entry steps

How does ERLIN1 contribute to cholesterol homeostasis and lipid metabolism?

ERLIN1's role in lipid metabolism is increasingly recognized:

• Phosphoinositide binding:

  • The erlin1/2 complex interacts selectively with monophosphorylated phosphoinositides

  • Strongest binding is to PI(3)P, approximately 5-fold greater than binding to PI(4)P and PI(5)P

  • This binding appears to be direct and independent of RNF170

• Cholesterol regulation:

  • ERLIN1 is implicated in cholesterol homeostasis

  • Studies have shown that ERLIN2 (partner of ERLIN1) supports cancer cell growth by regulating cytosolic lipid droplet production

  • The ERLIN1/2 scaffold restricts the secretory pathway by limiting cholesterol esterification

• Connection to sepsis and immune response:

  • ERLIN1 is differentially modulated among immune cells in response to cellular perturbations

  • During sepsis, ERLIN1 expression increases in whole-blood neutrophils and HL60 cell lines

  • This regulation has important implications for ER functions and/or ER membrane protein components during sepsis

  • Differential intracellular cholesterol dynamics were observed in HL60 cells upon LPS/PGN stimulation

How can I distinguish between ERLIN1 and ERLIN2 in experimental systems?

Distinguishing between these closely related proteins requires careful experimental design:

• Antibody selection:

  • Use specific antibodies that target unique regions of ERLIN1 to avoid cross-reactivity with ERLIN2

  • Be aware that pan-antibodies against both proteins may cross-react with unrelated proteins

  • When using immunoblotting, note that ERLIN1 (41 kDa) migrates slightly differently than ERLIN2 (43 kDa) on SDS-PAGE

• Knockout/knockdown strategies:

  • Generate specific knockouts for each protein using CRISPR/Cas9 technology targeting unique regions

  • For siRNA knockdown, use validated sequences specific to ERLIN1 (siErlin 1.5: CCACAAATAGGAGCAGCAT)

  • As control experiments, consider dual knockdown (siErlin 1&2: AGAAGCAATGGCCTGGTAC)

• Functional analysis approaches:

  • Assess IP3R ubiquitination and degradation, which are differentially affected by ERLIN1 vs. ERLIN2 knockout

  • In ERLIN1 KO cells, IP3R1 ubiquitination is inhibited by ~67%, while in ERLIN2 KO cells, it's inhibited by ~94%

  • ERLIN2 alone can bind activated IP3R1, while ERLIN1 alone cannot

What are the best experimental models for studying ERLIN1 function?

Several experimental models have proven effective for ERLIN1 studies:

• Cell line models:

  • αT3 cells: Effective for studying GnRH-induced, erlin1/2 complex-mediated IP3R1 ERAD

  • Huh7 cells: Suitable for studying ERLIN1's role in HCV infection

  • HL60 cells: Useful for investigating ERLIN1's role in immune response and sepsis

• Knockout/knockdown systems:

  • CRISPR/Cas9-mediated ERLIN1/2 double knockout (DKO) cells provide a clean background for reconstitution experiments

  • Single knockouts of either ERLIN1 or ERLIN2 allow assessment of their individual contributions

  • Reconstitution of knockouts with wild-type or mutant proteins to assess structure-function relationships

• Isolation techniques for in vitro studies:

  • Detergent-resistant membrane (DRM) isolation using Optiprep gradient flotation effectively concentrates erlin1/2 complexes

  • Immunoprecipitation with specific antibodies can isolate the native erlin1/2 complex and interacting partners

How can I investigate ERLIN1's interaction with the TMUB1-RNF170 complex?

Recent research has revealed the importance of ERLIN1 in scaffolding TMUB1 and RNF170:

• Co-immunoprecipitation approaches:

  • Use antibodies against ERLIN1, TMUB1, or RNF170 for reciprocal co-immunoprecipitation experiments

  • Note that ERLIN2 interacts specifically with the long isoform of TMUB1 (TMUB1-L), not the short isoform (TMUB1-S)

  • The interaction of TMUB1 with RNF170 is completely dependent on the presence of ERLIN scaffolds

• Structural domain mapping:

  • The N-terminal luminal domain of TMUB1-L is required for interaction with ERLINs

  • TMUB1-L and RNF170 have similar topology with a luminal domain, three transmembrane domains, and cytosolic domains

• Functional assays:

  • Analyze the distribution of TMUB1-L in detergent-resistant membrane (DRM) fractions, which is decreased in ERLIN1/2 double knockout cells

  • Reconstitution of DKO cells with wild-type ERLIN1/2 rescues this distribution

  • Mass spectrometry analysis of immunoprecipitated complexes can identify additional interaction partners in the complex