ATF6B Antibody

What is ATF6B and what cellular processes is it involved in?

ATF6B (activating transcription factor 6 beta) is an endoplasmic reticulum (ER)-transmembrane protein that plays a critical role in the ER stress response (ERSR). It functions as a regulator of ATF6α-mediated ER stress response through its repressor function. The protein is cleaved during ER stress, resulting in the translocation of its N-terminal fragment to the nucleus where it binds to ER stress-response elements (ERSE) in genes such as Bip/GRP78 .

ATF6B has five N-linked glycosylation sites, and this glycosylation is involved in ER stress-induced proteolysis that cleaves 110 kDa ATF6B to produce a nuclear form of 60 kDa ATF6B. This nuclear form acts as a repressor to ATF6α-mediated ERSR by binding to various ER stress-inducible promoters .

What applications are validated for ATF6B antibodies?

ATF6B antibodies have been validated for multiple research applications:

The antibody has been cited in numerous publications across different applications, including 15 publications for WB, 1 for IHC, and 3 for IF applications .

What are the recommended dilutions for different experimental applications?

The recommended dilutions vary by application and specific antibody. For the Proteintech ATF6B antibody (15794-1-AP), the following dilutions are recommended:

For other commercially available antibodies, such as Elabscience's E-AB-18330, the recommended dilutions are:

• WB: 1:500-1:2000

• IHC: 1:25-1:100

It is important to note that these are starting recommendations, and researchers should titrate the antibody in their specific experimental systems to obtain optimal results .

Why does ATF6B show different molecular weights in Western blots?

The calculated molecular weight of ATF6B is 77 kDa (703 amino acids), but the observed molecular weight in Western blots is typically around 110 kDa . This discrepancy can be attributed to several factors:

• Post-translational modifications, particularly glycosylation: ATF6B has five N-linked glycosylation sites that can increase the apparent molecular weight .

• Processing during ER stress: ATF6B is cleaved during the ER stress response, producing fragments of different sizes. The full-length protein is approximately 110 kDa, while the cleaved nuclear form is approximately 60-80 kDa .

• Different isoforms or splice variants: Various isoforms may be detected depending on the epitope recognized by the antibody.

If unexpected band sizes are observed, researchers should consider the possibility of different modified forms of the protein existing simultaneously, which can result in multiple bands on the membrane .

What are the essential controls for validating ATF6B antibody specificity?

To ensure antibody specificity for ATF6B, researchers should implement the following controls:

• Positive controls: Use verified positive samples such as Jurkat cells, human testis tissue, or HepG2 cells which are known to express ATF6B .

• Knockout/knockdown controls: Utilize ATF6B knockdown (KD) or knockout (KO) cell lines to confirm the specificity of bands observed in Western blots. Several publications have used this approach for validation .

• Peptide competition assay: Pre-incubate the antibody with the immunogenic peptide to demonstrate specific binding.

• Cross-reactivity testing: Test reactivity with closely related proteins, particularly ATF6α, to ensure the antibody specifically detects ATF6B and not other ATF family members.

• Multiple antibody validation: Use antibodies from different sources that recognize different epitopes of ATF6B to confirm consistent detection patterns.

How does ATF6B function differ from ATF6α in the ER stress response pathway?

ATF6B and ATF6α have distinct but interrelated functions in the ER stress response:

• Regulatory relationship: ATF6B functions as a repressor of ATF6α-mediated ER stress response. Through this repression of ATF6α, ATF6B activates the expression of many ERSR-related genes such as glucose-regulated protein 78 (GRP78) .

• Structural similarities and differences: Both ATF6B and ATF6α have conserved basic leucine-zipper and DNA binding domains but possess divergent transcriptional activation domains. These structural differences contribute to their distinct functions .

• Processing dynamics: Both proteins are cleaved during ER stress, but their processing rates and efficiency may differ, leading to temporal differences in their activation.

• Transcriptional targets: While they can bind to similar DNA elements, the gene sets they regulate may partially overlap but are not identical.

When ATF6 is silenced, there is increased IRE1 levels and XBP1 splicing, suggesting that ATF6 has an "off-switch" function for IRE1 signaling during ER stress . This indicates a complex interplay between the different branches of the unfolded protein response (UPR).

What methodological approaches can be used to study ATF6B cleavage during ER stress?

Studying ATF6B cleavage requires a combination of biochemical and imaging techniques:

• Western blotting with subcellular fractionation:

  • Separate nuclear and ER/cytoplasmic fractions

  • Use antibodies that recognize different domains (N-terminal vs. C-terminal) to track the cleaved fragments

  • Monitor the appearance of the 60 kDa nuclear form versus the 110 kDa full-length protein

• Fluorescence microscopy approaches:

  • Use immunofluorescence to track ATF6B localization before and during ER stress

  • Employ fluorescently tagged ATF6B constructs to monitor trafficking in live cells

  • Recommended dilution for IF/ICC: 1:50-1:500

• Inducing ER stress experimentally:

  • Chemical inducers: thapsigargin (Tg), tunicamycin (Tm), or brefeldin A (BFA)

  • Time-course experiments to capture the kinetics of cleavage

  • Monitor activation of other UPR branches simultaneously (IRE1, PERK)

• Site-directed mutagenesis:

  • Create glycosylation site mutants to study the role of glycosylation in ATF6B cleavage

  • A fully unglycosylated mutant of ATF6B has been shown not to be cleaved, leading to modulations of ERSR gene expression

What is the evidence linking ATF6B polymorphisms to respiratory function and lung diseases?

There is emerging evidence connecting ATF6B to lung function:

• A genome-wide association study identified a chromosomal region including ATF6B that is associated with lung function parameters, specifically the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) .

• Polymorphisms of ATF6B are potentially associated with FEV1 decline by aspirin provocation in patients with aspirin-exacerbated respiratory disease (AERD) .

• The study investigated four common single nucleotide polymorphisms (SNPs) of ATF6B in 93 AERD patients and 96 aspirin-tolerant asthma (ATA) controls. The results suggested possible associations between specific genetic variants and respiratory function .

• ER stress has been observed to activate NF-kappaB and induce inflammatory responses that are implicated in asthma, providing a potential mechanistic link between ATF6B function and respiratory diseases .

These findings suggest that ATF6B may play an important role in respiratory function, potentially through its involvement in ER stress responses in lung tissue.

What antigen retrieval methods are recommended for ATF6B immunohistochemistry?

For optimal ATF6B detection in immunohistochemistry applications, the following antigen retrieval methods are recommended:

• Primary recommendation: TE buffer pH 9.0

  • This is the suggested method for ATF6B detection in human brain tissue and other samples

• Alternative method: Citrate buffer pH 6.0

  • Can be used as an alternative approach if the primary method doesn't yield satisfactory results

The appropriate antigen retrieval method can significantly impact the sensitivity and specificity of ATF6B detection, particularly in formalin-fixed, paraffin-embedded (FFPE) tissues. Verification using positive control tissues such as human brain tissue or human colorectal cancer is recommended .

How can researchers effectively study the interaction between ATF6B and ATF6α?

To investigate the interaction and regulatory relationship between ATF6B and ATF6α, researchers can employ several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use ATF6B antibody for immunoprecipitation (recommended amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Western blot for ATF6α in the precipitate

  • Perform the reciprocal experiment using ATF6α antibody for IP and blotting for ATF6B

• Chromatin immunoprecipitation (ChIP):

  • Determine if ATF6B and ATF6α bind to the same or different promoter regions

  • Identify potential competitive binding to shared DNA elements

• Expression modulation studies:

  • Overexpress the N-terminal transcriptionally active domain of ATF6B (similar to the approach used with ATF6α(1-373) in )

  • Silence ATF6B using shRNA or siRNA and examine effects on ATF6α activity

  • Create double knockdown models to study synergistic effects

• Proximity ligation assay (PLA):

  • Visualize and quantify protein-protein interactions in situ

  • Determine subcellular locations where ATF6B and ATF6α interact

• Reporter gene assays:

  • Use ERSE-containing promoter constructs

  • Test the effects of ATF6B and ATF6α individually and in combination

These approaches can help elucidate the complex regulatory relationship between these two transcription factors in the ER stress response.

What are the considerations for selecting appropriate control cell lines and tissues for ATF6B experiments?

Selection of appropriate controls is critical for ATF6B research. Based on validation data, researchers should consider:

Cell lines with confirmed ATF6B expression:

• Jurkat cells: Validated for WB and IP applications

• HepG2 cells: Validated for IF/ICC applications

• HeLa cells: Used in multiple ATF6B antibody validations

Tissue samples with confirmed expression:

• Human testis tissue: Positive in WB applications

• Human brain tissue: Validated for IHC applications

• Human colorectal cancer: Validated for IHC applications

Negative controls to consider:

• Cell lines with ATF6B knockdown or knockout

• Tissues from ATF6B knockout animal models, if available

• Isotype controls for immunostaining (Rabbit IgG for polyclonal antibodies)

When designing experiments, researchers should also consider:

• Expression levels may vary with cell stress status

• ATF6B expression may be induced during ER stress conditions

• Cell type-specific processing and post-translational modifications

How does ATF6B contribute to the integrated stress response and what methodologies can measure this involvement?

ATF6B plays a role in the integrated stress response (ISR), particularly through its interaction with the unfolded protein response (UPR). To study this involvement:

• Integrated stress response monitoring:

  • Measure phosphorylation of eIF2α, a convergence point for various stress signals

  • Assess expression of downstream targets like ATF4 and CHOP

  • Use reporter systems to monitor branch-specific activation of the UPR (IRE1, PERK, and ATF6 pathways)

• Multi-parametric analysis:

  • Simultaneously monitor ATF6B cleavage, XBP1 splicing (IRE1 pathway), and PERK activation

  • Perform time-course experiments to understand the temporal relationship between ATF6B activity and other ISR components

  • High-content live cell imaging can be particularly valuable for this purpose

• Cross-talk investigation:

  • Overexpression of ATF6B N-terminal domain (similar to the ATF6(1-373) approach) can help determine effects on other UPR branches

  • shRNA-mediated silencing of ATF6B followed by measurement of IRE1 and PERK activity

  • qPCR analysis of UPR target genes in ATF6B-modulated systems

• Cell death and survival analysis:

  • Monitor cellular outcomes (survival vs. apoptosis) in relation to ATF6B activity

  • Use flow cytometry with propidium iodide staining to assess cell death rates

  • Compare outcomes between wildtype and ATF6B-modulated cells under various stress conditions

Understanding the role of ATF6B in the ISR is important as it has implications for diseases where ER stress plays a pathogenic role.

What are the latest techniques for studying ATF6B involvement in glycosylation-dependent cellular processes?

ATF6B function is modulated by its glycosylation status, making this an important area of investigation:

• Glycosylation site mutation analysis:

  • Create single or multiple glycosylation site mutants using site-directed mutagenesis

  • Compare the processing, localization, and activity of wild-type vs. glycosylation-deficient ATF6B

  • Fully unglycosylated ATF6B mutants have been shown not to undergo cleavage, affecting ERSR gene expression

• Glycosylation status detection:

  • Use PNGase F or Endo H treatment followed by Western blotting to distinguish between glycosylated and non-glycosylated forms

  • Mobility shift assays to track glycosylation changes during ER stress

  • Glycoprotein-specific staining methods in combination with IF/ICC (dilution 1:50-1:500)

• Mass spectrometry approaches:

  • Identify specific glycosylation patterns on ATF6B

  • Compare glycosylation profiles under normal vs. stress conditions

  • Map the glycosylation sites that are critical for ATF6B function

• Pulse-chase experiments:

  • Track the kinetics of ATF6B glycosylation, processing, and degradation

  • Compare processing rates between wild-type and glycosylation mutants

  • Determine half-life differences between glycosylated and non-glycosylated forms

Understanding the role of glycosylation in ATF6B function can provide insights into how post-translational modifications regulate the ER stress response.

How can researchers investigate the role of ATF6B in conditions associated with retinal function and development?

Recent research has linked ATF6 (the alpha isoform) to retinal function, specifically to achromatopsia and cone dysfunction . To investigate whether ATF6B has similar roles:

• Expression analysis in retinal tissues:

  • Immunohistochemistry of human or animal retinal sections

  • Layer-specific analysis to identify cell types expressing ATF6B

  • Developmental time-course of ATF6B expression

• Functional studies in retinal cells:

  • ATF6B knockdown or overexpression in retinal cell cultures

  • Assessment of cone photoreceptor development and function

  • Electrophysiological measurements to detect functional changes

• Genetic association studies:

  • Screen for ATF6B variants in patients with unexplained retinal disorders

  • Similar to the approach that identified ATF6 mutations in achromatopsia

  • Functional characterization of any identified variants

• In vivo models:

  • Conditional knockout or knockdown of ATF6B in retinal cells

  • Phenotypic analysis focusing on visual function and retinal development

  • Comparison with ATF6α knockout models to identify unique vs. redundant functions

• ER stress induction in retinal tissues:

  • Compare ATF6B activation in healthy vs. diseased retinal samples

  • Examine the UPR in models of retinal degeneration

  • Test if modulation of ATF6B activity affects disease progression

These approaches can help determine whether ATF6B plays a role in retinal function similar to or distinct from that of ATF6α, which has been linked to achromatopsia through mutations that cause cone dysfunction .