mxi1 Antibody

What is MXI1 and what are its primary biological functions?

MXI1 is a transcriptional repressor that belongs to the Mad family of proteins, functioning as a negative regulator of c-Myc oncogenic activity. It plays crucial roles in cell growth regulation and differentiation by forming heterodimers with Max protein, which is essential for Myc oncogene function . MXI1 binds to E-box sequences in DNA and recruits Sin3 co-repressor complexes to inhibit transcription of Myc target genes. This regulatory mechanism helps maintain proper control of cell proliferation and differentiation pathways, with implications for both normal development and disease states .

What are the recognized isoforms of MXI1, and how do they differ?

Two major isoforms of MXI1 have been identified: MXI1 (also referred to as MXI1-1) and MXI1-0. MXI1-0 is alternatively transcribed from an upstream exon (exon 0), resulting in different amino-terminal sequences compared to the canonical MXI1, though both share identical Max- and DNA-binding domains . Functionally, these isoforms differ significantly: while both can bind Max and recognize E-box binding sites, MXI1-0 fails to repress c-Myc-dependent transcription, unlike MXI1 . Additionally, MXI1-0 is predominantly localized in the cytoplasm, whereas MXI1-1 exhibits primarily nuclear localization .

How do available MXI1 antibodies differ in their specificity and applications?

Available MXI1 antibodies vary in their host species, clonality, and application suitability:

• Mxi1 Antibody (MXI1C2a) is a mouse monoclonal IgG1 kappa antibody that detects human MXI1 protein specifically in western blotting (WB) and immunoprecipitation (IP) applications .

• MXI1 Rabbit Polyclonal Antibody (CAB12098) is derived from rabbits and shows broader species reactivity, detecting MXI1 in human, mouse, and rat samples. It is recommended for Western blot and ELISA applications at dilutions of 1:500-1:2000 .

The choice between these antibodies depends on experimental needs, including required specificity, application method, and target species.

How does the expression of MXI1 isoforms change under hypoxic conditions, and what are the implications?

Under hypoxic conditions, MXI1 expression is significantly upregulated in pulmonary arterial smooth muscle cells (PASMCs) but not in pulmonary arterial endothelial cells (PAECs) . This upregulation follows a time-dependent pattern, with expression peaking after 12 hours of hypoxia exposure . Notably, MXI1-0 has been identified as the predominant isoform induced by hypoxia .

This differential expression has significant implications for understanding hypoxia-related pathologies. For example, in hypoxic pulmonary hypertension (HPH), MXI1-0 promotes pathogenesis through MEK/ERK/c-Myc-mediated proliferation of PASMCs . Since MXI1-0, unlike MXI1, cannot antagonize c-Myc activity, its increased expression under hypoxia may contribute to dysfunctional cell growth control in hypoxic environments.

What signaling pathways does MXI1-0 interact with, and how do these differ from canonical MXI1?

MXI1-0 interacts with the MEK/ERK signaling pathway to promote the expression of the proto-oncogene c-Myc, subsequently enhancing cell proliferation . This stands in contrast to canonical MXI1, which functions as a transcriptional repressor of c-Myc-dependent gene expression .

Research has demonstrated that inhibitors targeting this pathway, including the MEK inhibitor PD98059 and the c-Myc inhibitor 10058F4, can counteract MXI1-0-induced cell proliferation . These findings suggest a potential therapeutic approach for conditions where MXI1-0 overexpression contributes to pathogenesis, such as in hypoxic pulmonary hypertension and possibly certain cancers.

How is MXI1 expression altered in pathological conditions, and what is the differential role of its isoforms?

MXI1 expression patterns change significantly in several pathological conditions. For instance, the relative levels of MXI1-0 are higher in primary glioblastoma tumors compared to normal brain tissue . Similarly, MXI1 is significantly upregulated in the PASMCs of hypoxic pulmonary hypertension (HPH) patients .

The differential expression of MXI1 isoforms can have profound implications for disease progression. While canonical MXI1 generally functions as a tumor suppressor through its ability to antagonize c-Myc activity, MXI1-0 may actually promote cell proliferation and potentially contribute to tumor growth or vascular remodeling in HPH . This functional dichotomy suggests that the balance between MXI1 isoforms, rather than total MXI1 expression, may be critical in determining disease outcomes.

What are the optimal protocols for detecting MXI1 isoforms in Western blotting experiments?

For optimal detection of MXI1 isoforms via Western blotting, researchers should consider the following methodological approach:

• Sample preparation: For cell lysates, exposure to specific conditions (e.g., hypoxia for 12 hours) may be necessary to detect induced isoforms .

• Antibody selection:

  • For detection of all isoforms, use a pan-MXI1 antibody

  • For isoform-specific detection, consider using epitope-tagged constructs (HA-tagged MXI1-0 or Flag-tagged MXI1-1) and corresponding tag antibodies

• Molecular weight expectations: Different isoforms will appear at distinct molecular weights; a 45 kDa band has been observed for MXI1 in clinical pulmonary specimens .

• Recommended antibody dilutions: For the MXI1 Rabbit Polyclonal Antibody (CAB12098), a dilution range of 1:500-1:2000 is recommended for Western blotting applications .

• Positive controls: Consider using lysates from U-87MG, A-549, HepG2, mouse brain, rat heart, or rat brain tissues, which have been validated as positive samples for MXI1 detection .

How can researchers differentiate between MXI1 isoforms in experimental systems?

Differentiating between MXI1 isoforms requires specific experimental strategies:

• RT-PCR with isoform-specific primers: Design forward primers specific to exon 0 (for MXI1-0) and exon 1 (for MXI1), with a common reverse primer from a shared exon. This approach allows for multiplex one-step RT-PCR detection of both isoforms simultaneously .

• Epitope tagging: Generate expression constructs with different tags for each isoform (e.g., HA-tagged MXI1-0 and Flag-tagged MXI1-1) to distinguish them in overexpression experiments .

• Subcellular localization studies: Immunofluorescence can be used to distinguish isoforms based on their predominant localization patterns (MXI1-0 in both cytoplasm and nucleus, MXI1-1 primarily in the nucleus) .

• Functional assays: Since MXI1-0 fails to repress c-Myc-dependent transcription while MXI1 does, reporter assays measuring c-Myc-dependent transcriptional activity can help differentiate the functional presence of these isoforms .

What considerations should be made when selecting positive and negative controls for MXI1 antibody validation?

When validating MXI1 antibodies, careful selection of controls is essential:

Positive controls:

• Validated cell lines: U-87MG, A-549, HepG2 cells have been confirmed to express detectable levels of MXI1

• Tissue samples: Mouse brain, rat heart, and rat brain tissues are suitable positive controls

• Overexpression systems: Cells transfected with MXI1 expression constructs (preferably tagged versions for additional verification)

Negative controls:

• siRNA or shRNA knockdown: Cells treated with MXI1-specific siRNA should show reduced signal

• Cell types with minimal MXI1 expression: Identification of low-expressing cell lines through literature review

• Blocking peptide controls: Pre-incubation of the antibody with immunizing peptide should abolish specific signals

Additional validation methods:

• Comparison of results with multiple antibodies targeting different epitopes

• Parallel detection using complementary methods (e.g., mRNA quantification)

• Cross-species validation when using antibodies with reported multi-species reactivity

Why might researchers observe different molecular weights for MXI1 on Western blots?

Researchers may observe variations in MXI1 molecular weights on Western blots due to several factors:

• Isoform detection: MXI1-0 and MXI1-1 have different amino-terminal sequences resulting in distinct molecular weights .

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can alter protein migration patterns.

• Antibody specificity: Different antibodies may recognize specific regions or epitopes, leading to detection of distinct isoforms or modified forms.

• Species variations: Human MXI1 isoforms have slight differences from mouse MXI1 variants, particularly in C-terminal residues .

• Sample preparation conditions: Denaturing conditions, reducing agents, and buffer compositions can affect protein migration.

In published research, a pan-MXI1 antibody detected a 45 kD protein in clinical pulmonary specimens and hypoxic PASMCs , while the calculated molecular weight is approximately 26 kDa . This discrepancy could be due to post-translational modifications or the specific isoform being detected.

How can discrepancies in MXI1 expression between different experimental systems be reconciled?

Discrepancies in MXI1 expression across experimental systems can be addressed through systematic analysis:

• Cell type-specific regulation: MXI1 expression varies significantly between cell types. For example, hypoxia induces MXI1 in PASMCs but not in PAECs . Document and compare cell types used across studies.

• Isoform-specific detection: Ensure methods are capturing the same isoforms. MXI1-0 and MXI1-1 may show different expression patterns and responses to stimuli .

• Environmental conditions: Standardize experimental conditions, as factors like hypoxia duration significantly impact MXI1 expression .

• Antibody characteristics: Different antibodies may have varying affinities for MXI1 isoforms or epitopes. Use multiple antibodies and complementary detection methods.

• Quantification methods: Standardize protein loading controls and quantification methods across experiments.

• Subcellular fractionation effects: Since MXI1 isoforms localize differently (MXI1-0 in cytoplasm and nucleus; MXI1-1 primarily nuclear) , whole-cell versus nuclear extracts may yield different results.

What factors might affect antibody performance in detecting endogenous versus overexpressed MXI1?

Several factors can influence antibody performance when comparing detection of endogenous versus overexpressed MXI1:

• Expression level disparities: Overexpression systems typically produce protein levels far exceeding endogenous expression, which may:

  • Saturate antibody binding sites

  • Lead to non-physiological aggregate formation

  • Cause aberrant localization patterns

• Epitope accessibility: Tag addition in overexpression constructs may alter protein folding or expose/mask epitopes recognized by anti-MXI1 antibodies.

• Isoform representation: Overexpression typically involves a single isoform, while endogenous detection may capture multiple isoforms in varying ratios .

• Post-translational modifications: Overexpressed proteins may undergo different post-translational modifications compared to endogenous proteins.

• Background interference: High expression levels from overexpression may overcome background issues that confound detection of lower-abundance endogenous protein.

To address these issues, researchers should:

• Use both tag-specific and MXI1-specific antibodies when working with tagged constructs

• Include appropriate controls (untransfected cells, empty vector controls)

• Titrate expression plasmid amounts to achieve more physiological expression levels

• Compare multiple antibodies with different epitope specificities

How can MXI1 antibodies be utilized to investigate the Myc/Max/Mad regulatory network in cancer research?

MXI1 antibodies provide valuable tools for investigating the Myc/Max/Mad regulatory network in cancer research through multiple approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation experiments using MXI1 antibodies can identify binding partners and complexes

  • Proximity ligation assays can visualize MXI1-Max interactions in situ

  • Pull-down assays can assess how mutations or drug treatments affect interaction dynamics

• Chromatin studies:

  • Chromatin immunoprecipitation (ChIP) with MXI1 antibodies can map genomic binding sites

  • Sequential ChIP (ChIP-reChIP) can determine co-occupancy with Max or other factors

  • ChIP-seq analysis can reveal genome-wide binding patterns in different cancer contexts

• Expression profiling:

  • Immunohistochemistry using MXI1 antibodies on tissue microarrays can assess expression across tumor types and grades

  • Correlation of MXI1 isoform levels with c-Myc expression can provide prognostic insights

  • Western blot analysis of nuclear vs. cytoplasmic fractions can determine MXI1 isoform distribution

• Functional studies:

  • Visualization of MXI1 localization changes in response to signaling events

  • Assessment of MXI1 binding to target promoters after therapeutic interventions

  • Monitoring changes in MXI1/MXI1-0 ratio during tumor progression

What methodological approaches can detect the interaction between MXI1 isoforms and the MEK/ERK pathway?

To investigate interactions between MXI1 isoforms and the MEK/ERK pathway, researchers can employ the following methodological approaches:

• Phosphorylation state analysis:

  • Western blotting with phospho-specific antibodies to detect ERK activation in response to MXI1 isoform overexpression or knockdown

  • Immunoprecipitation of MXI1 followed by phospho-specific antibody detection to determine if MXI1 itself is phosphorylated by ERK

• Pathway perturbation:

  • Pharmacological inhibition using MEK inhibitors (e.g., PD98059) to determine effects on MXI1-0-induced cellular responses

  • siRNA-mediated knockdown of pathway components to establish functional relationships

  • Constitutively active or dominant-negative ERK constructs to manipulate pathway activity

• Protein-protein interaction studies:

  • Co-immunoprecipitation using MXI1 antibodies followed by detection of MEK/ERK components

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET approaches with fluorescently tagged proteins to monitor interactions in live cells

• Transcriptional regulation analysis:

  • Reporter assays to measure c-Myc-dependent transcription in response to pathway modulation

  • ChIP-qPCR to assess MXI1 binding to target genes following MEK/ERK inhibition

  • RNA-seq analysis comparing MXI1-0 overexpression with and without MEK inhibition

• In vivo validation:

  • Animal models with MEK/ERK pathway inhibition to confirm effects on MXI1-mediated pathologies such as HPH

How might researchers apply MXI1 antibodies in studying hypoxia-related pathologies?

For studying hypoxia-related pathologies, MXI1 antibodies can be applied in several sophisticated research approaches:

• Expression dynamics analysis:

  • Time-course studies of MXI1 isoform induction under varying degrees of hypoxia

  • Cell type-specific expression patterns (e.g., comparing PASMCs vs. PAECs)

  • Correlation of MXI1-0:MXI1 ratio with disease severity markers

• Signaling pathway integration:

  • Investigation of HIF-1α regulation of MXI1 transcription

  • Analysis of how MXI1-0 activates MEK/ERK signaling under hypoxic conditions

  • Assessment of c-Myc target gene expression in response to hypoxia-induced MXI1-0

• Therapeutic target validation:

  • Immunohistochemical assessment of MXI1 expression in patient samples with hypoxia-related pathologies

  • Monitoring MXI1 isoform changes in response to therapeutic interventions

  • Correlation of treatment efficacy with MXI1-0 expression reduction

• Subcellular dynamics:

  • Fractionation studies combined with Western blotting to track isoform-specific localization changes during hypoxia

  • Immunofluorescence to visualize MXI1-0 translocation under hypoxic conditions

  • Co-localization studies with oxygen-sensing pathway components

• Preclinical model applications:

  • Validation of MXI1 targeting in animal models of hypoxic pulmonary hypertension

  • Immunohistochemical assessment of vascular remodeling in relation to MXI1 expression

  • Monitoring MXI1 isoform ratios as biomarkers of disease progression and treatment response