NFE2L1 Monoclonal Antibody

What is NFE2L1 and what cellular functions does it regulate?

NFE2L1, also known as Nrf1, functions as an endoplasmic reticulum membrane sensor that translocates to the nucleus in response to various cellular stresses, where it acts as a transcription factor . It plays essential roles in multiple biological processes including redox signaling, cellular metabolism, proteasome homeostasis, and metabolic regulation . NFE2L1 constitutes a precursor of the transcription factor NRF1 and acts as a key sensor of cellular stresses such as cholesterol excess, oxidative stress, and proteasome inhibition . The protein is released from the endoplasmic reticulum membrane following cleavage by the protease DDI2, allowing nuclear translocation where it regulates expression of protective genes . Notably, NFE2L1 knockout in mice leads to embryonic lethality, indicating its essential function in development .

How do NFE2L1 monoclonal antibodies perform in Western blot applications?

NFE2L1 monoclonal antibodies, such as the rabbit recombinant monoclonal antibody [EPR25225-16] (ab302520), demonstrate high specificity in Western blot applications when properly optimized . When using this antibody at a 1/1000 dilution with 5% NFDM/TBST as blocking buffer, researchers can detect NFE2L1 as a band at approximately 140 kDa in wild-type cells . Importantly, validation experiments show no signal at this size in NFE2L1 knockout cell lines, confirming antibody specificity . For optimal results, high-sensitivity ECL substrates capable of detecting proteins in the mid-femtogram range are recommended, particularly when working with low-abundance isoforms . Researchers should anticipate that proteasome inhibition (e.g., with MG-132 treatment) can increase detectable levels of NFE2L1, which serves as a positive control for antibody performance .

What are the major structural domains of NFE2L1 and how do they relate to function?

NFE2L1 protein contains eight primary functional structural domains that determine its cellular localization and activity:

The NFE2L1 protein anchors to the endoplasmic reticulum membrane through the NTD, Neh6L, and CTD domains, positioning the AD1, NST, AD2, and SR regions in the ER lumen while the DBD domain remains in the cytosol . This arrangement is crucial for NFE2L1's function as a stress sensor that can be released from the ER upon appropriate signals.

How should researchers design experiments to investigate NFE2L1's response to metabolic stress?

When investigating NFE2L1's response to metabolic stress, researchers should consider a multi-faceted experimental approach. Begin by testing how NFE2L1 protein levels and electrophoretic mobility change in response to varying glucose and serum concentrations . Western blot analysis should be performed with cells cultured under different conditions: normal glucose (typically 25mM), glucose deprivation, with and without serum . Monitor both the total NFE2L1 protein levels and any shifts in band position on SDS-PAGE that might indicate post-translational modifications, especially glycosylation .

Additionally, investigate associated signaling pathways by examining the phosphorylation status of AMPK and mTOR, which show differential responses to glucose and serum conditions . Include metabolic stress inducers such as proteasome inhibitors (e.g., MG-132, 10μM for 6 hours) to observe how NFE2L1 responds . For comprehensive analysis, pair these protein-level investigations with metabolomic profiling, seahorse analysis for mitochondrial function, and transcriptome analysis to reveal how NFE2L1 regulates glucose metabolism pathways .

What controls are essential when validating NFE2L1 antibody specificity?

Proper validation of NFE2L1 antibody specificity requires several critical controls:

• Genetic knockout controls: Compare antibody reactivity in wild-type versus NFE2L1 knockout cells (e.g., NFE2L1 knockout HEK-293T cell line) . A specific antibody should detect bands at the expected molecular weight in wild-type samples but show no signal in knockout samples.

• siRNA knockdown controls: Test antibody reactivity in cells transfected with NFE2L1-specific siRNA versus scrambled siRNA control . This demonstrates specificity through reduced signal intensity corresponding to the degree of knockdown.

• Treatment controls: Include samples treated with proteasome inhibitors (e.g., MG-132) which typically increase NFE2L1 levels . This serves as a positive control for antibody detection sensitivity.

• Cross-reactivity assessment: Test the antibody against related proteins, particularly NFE2L2 (Nrf2) which shares structural similarities with NFE2L1, to ensure the antibody doesn't cross-react.

• Multiple detection methods: When possible, validate findings using orthogonal techniques such as immunofluorescence or mass spectrometry to confirm specificity.

These controls collectively provide strong evidence for antibody specificity and reliability in experimental applications.

How can researchers distinguish between different NFE2L1 isoforms using monoclonal antibodies?

Distinguishing between NFE2L1 isoforms requires careful antibody selection and experimental design. NFE2L1 exists in several isoforms, including the full-length protein and variants like NFE2L1-616, which lacks the ER targeting domain present in other isoforms . To differentiate between these isoforms:

• Select antibodies with mapped epitopes: Choose monoclonal antibodies with epitopes that either recognize all isoforms (common regions) or specifically target unique regions of particular isoforms. For example, antibodies targeting the N-terminal domain will not detect NFE2L1-616, which has a distinct first exon .

• Use molecular weight discrimination: Different isoforms have distinct molecular weights on SDS-PAGE. NFE2L1-616 is approximately 10 kDa smaller than full-length NFE2L1 due to the absence of 152 amino acids at the C-terminus . When using a high-resolution gel system, carefully analyze band patterns at 140 kDa (full-length) versus lower molecular weights.

• Combine with subcellular fractionation: NFE2L1-616 constitutively localizes in the nucleus while other isoforms are initially ER-bound . Perform nuclear/cytoplasmic/ER fractionation before immunoblotting to help identify specific isoforms based on their predominant localization.

• Employ isoform-specific knockdown: Use siRNAs targeting specific exons to selectively deplete certain isoforms and confirm antibody specificity for each variant.

How do researchers investigate NFE2L1's dual roles in transcription-dependent and independent functions?

NFE2L1 exhibits both transcription factor-dependent and independent functions, requiring specialized approaches to differentiate between these roles . To investigate this dual functionality:

• Domain-specific mutants: Create truncated NFE2L1 constructs, such as NFE2L1 ΔC (lacking the DNA binding domain), which retains non-transcriptional activities while losing transcription factor function . Compare the effects of full-length versus truncated expression on various cellular processes.

• Transcriptomic analysis: Perform RNA-seq comparing cells expressing wild-type NFE2L1, transcriptionally inactive mutants, and NFE2L1 knockout cells to identify genes regulated through transcription-dependent versus independent mechanisms .

• Pathway-specific assays: Measure specific readouts for pathways regulated by NFE2L1. For example, assess proteasome activity, redox homeostasis, and immune responses (predominantly transcription factor-dependent) versus metabolism, ribosome function, and Wnt/β-catenin signaling (which may involve non-transcriptional mechanisms) .

• Subcellular localization studies: Use fluorescently tagged NFE2L1 constructs to track localization, comparing wild-type protein (which can translocate between ER and nucleus) with mutants confined to specific compartments to correlate localization with distinct functions.

• Protein-protein interaction studies: Perform co-immunoprecipitation with wild-type and domain-specific mutants to identify interaction partners that mediate transcription-independent functions.

Why might NFE2L1 antibodies detect multiple bands on Western blots and how should these be interpreted?

Multiple bands on Western blots with NFE2L1 antibodies are common and can be attributed to several factors that researchers should interpret carefully:

• Protein processing and cleavage: NFE2L1 undergoes proteolytic processing by DDI2 protease to release it from the ER membrane . This generates fragments of different sizes that may be detected as distinct bands.

• Isoforms: The NFE2L1 gene encodes several protein isoforms, including full-length variants (like TCF11 and Nrf1a) and shorter variants like NFE2L1-616 . These would appear as bands of different molecular weights.

• Post-translational modifications: NFE2L1 undergoes extensive post-translational modifications, including glycosylation, which can create bands with altered mobility . Glycosylated NFE2L1 appears as a higher molecular weight band compared to unmodified protein.

• Degradation products: As NFE2L1 is involved in the proteasomal pathway, it is subject to regulated degradation. Partial degradation products may appear as lower molecular weight bands.

To interpret these multiple bands correctly:

• Compare with appropriate controls (knockout/knockdown cells)

• Use treatments that affect specific modifications (e.g., glycosylation inhibitors)

• Perform subcellular fractionation to correlate band patterns with localization

• Consider using isoform-specific antibodies when available

What are the optimal conditions for detecting NFE2L1 in Western blot applications?

Based on validated protocols, the following conditions provide optimal results for NFE2L1 detection by Western blot :

For enhanced detection of specific NFE2L1 forms, consider these additional optimizations:

• Use gradient gels (4-15%) for better separation of high molecular weight proteins

• Include phosphatase inhibitors in lysis buffer if studying phosphorylation

• For glycosylated forms, compare samples with and without treatment with endoglycosidases

• When studying stress responses, include paired samples with and without stressors (oxidative stress, proteasome inhibition, or glucose deprivation)

How can NFE2L1 antibodies be utilized to study neurodegenerative disease mechanisms?

NFE2L1 plays a neuroprotective role through regulation of proteasomal function, making it a valuable target for neurodegenerative disease research . When investigating NFE2L1 in this context:

• Proteasome dysfunction models: Use NFE2L1 antibodies to monitor expression and activation in cellular models treated with proteasome inhibitors or expressing neurodegenerative disease-associated proteins (e.g., mutant tau, α-synuclein, or huntingtin) . Compare NFE2L1 levels and localization between normal and diseased states.

• Brain tissue analysis: Apply immunohistochemistry with NFE2L1 antibodies to analyze expression patterns in brain regions affected by neurodegeneration, comparing control and disease tissues. Look for correlations between NFE2L1 expression/localization and markers of proteasomal dysfunction or protein aggregation.

• NFE2L1 activation therapy: When testing compounds designed to upregulate NFE2L1 as potential therapeutics, use antibodies to confirm target engagement and measure downstream effects on proteasome subunit expression . Monitor both total NFE2L1 levels and nuclear translocation as markers of activation.

• Cell-type specific analysis: Combine NFE2L1 antibodies with neuronal, astrocyte, or microglial markers in immunofluorescence studies to determine cell-type specific expression patterns and responses to stressors.

• Subcellular tracking: Use fractionation followed by immunoblotting to track NFE2L1 translocation from ER to nucleus in response to proteotoxic stress, which is often impaired in neurodegenerative conditions.

How should researchers approach investigating NFE2L1's role in metabolic disorders?

NFE2L1 has been implicated in various metabolic disorders including obesity, diabetes, and non-alcoholic steatohepatitis (NASH) . When studying NFE2L1 in metabolic contexts:

• Glucose sensing mechanism: Investigate NFE2L1's role as a glucose sensor by examining how its glycosylation status changes under various glucose concentrations. Compare wild-type cells with NFE2L1-deficient cells to assess differences in glucose uptake and metabolism .

• Tissue-specific analyses: Different metabolic phenotypes emerge from NFE2L1 dysfunction in distinct tissues - hyperinsulinemia and glucose intolerance in pancreatic β cells, adipocyte hypertrophy in fat tissue, and NASH in liver . Design tissue-specific experiments with appropriate controls for each context.

• Metabolic reprogramming: Use NFE2L1 antibodies alongside metabolic pathway markers to investigate how NFE2L1 deficiency reprograms glucose metabolism, particularly focusing on the Warburg effect in hepatic cells and mitochondrial function .

• Energy sensor interconnection: Examine the relationship between NFE2L1 and other energy sensors by monitoring how NFE2L1 levels correlate with AMPK and mTOR phosphorylation under various nutrient conditions .

• Intervention studies: When testing therapeutic interventions targeting NFE2L1 in metabolic disorders, use antibodies to confirm that the intervention successfully modulates NFE2L1 expression or activity before assessing metabolic endpoints.