MAGOH Antibody

What is MAGOH and what cellular functions does it perform?

MAGOH belongs to the mago nashi family and serves as a core component of the splicing-dependent multiprotein exon junction complex (EJC). The EJC is a dynamic structure consisting of core proteins and peripheral nuclear and cytoplasmic factors that associate transiently during assembly or subsequent mRNA metabolism .

MAGOH functions include:

• Pre-mRNA splicing as a component of the spliceosome

• Marking exon-exon junctions in mature mRNA

• Regulating nuclear mRNA export

• Influencing subcellular mRNA localization

• Affecting translation efficiency

• Participating in nonsense-mediated mRNA decay (NMD)

The MAGOH-RBM8A heterodimer inhibits EIF4A3's ATPase activity, trapping ATP-bound EJC core onto spliced mRNA. This heterodimer also interacts with PYM1, leading to EJC disassembly in the cytoplasm and enhancing translation of EJC-bearing spliced mRNAs .

How do MAGOH and MAGOHB differ in function and expression?

MAGOH and MAGOHB are paralogues with highly similar protein sequences. Their functional relationship includes:

• Both proteins play redundant roles as core components of the EJC and in the NMD pathway

• Targeting both genes via siRNAs is required for complete knockdown of MAGOH proteins

• They compete with each other for cellular interaction partners, as demonstrated by co-immunoprecipitation experiments where expression of FLAG-MAGOH decreased with increasing amounts of transfected V5-MAGOHB and vice versa

• Despite redundancy, individual knockdown experiments reveal distinct phenotypic consequences, suggesting some functional specialization

Expression patterns vary across tissues, with differential expression observed in healthy versus cancerous tissues .

What applications are MAGOH antibodies suitable for?

Based on validated applications from multiple sources, MAGOH antibodies show utility in:

For optimal results, each reagent should be titrated in your specific testing system .

What is the best method to validate MAGOH antibody specificity?

To validate MAGOH antibody specificity, implement a multi-step approach:

• Cross-reactivity assessment: Determine whether the antibody recognizes MAGOH, MAGOHB, or both. For example, ab180505 was shown to cross-react with both human MAGOH and MAGOHB in dot blot analysis .

• Molecular weight verification: Confirm band detection at the expected molecular weight of 17 kDa (both calculated and observed) .

• Positive control selection: Use cells/tissues with known MAGOH expression. Validated positive controls include:

  • For WB: K-562, HeLa, HL-60, Raji, LNCaP, HEK-293, and Jurkat cells

  • For IHC: Human ovary tumor tissue and endometrial adenocarcinoma tissue

  • For IP: K-562 cells

• Knockdown validation: Perform siRNA-mediated knockdown of MAGOH/MAGOHB and confirm reduction in antibody signal. Note that targeting both MAGOH genes is required for complete protein knockdown .

• Antigen competition: Perform pre-absorption with the specific immunogen (e.g., MAGOH fusion protein Ag3004) .

What lysate preparation methods are optimal for MAGOH antibody detection in Western blot?

For optimal MAGOH detection in Western blotting:

• Buffer selection: Use PBS with protease inhibitors for initial cell lysis.

• Sample preparation:

  • Load 20 μg of total protein per lane as demonstrated in successful WB applications

  • Denature samples in standard Laemmli buffer at 95°C for 5 minutes

• Blocking conditions:

  • Use 5% non-fat dry milk in TBST as demonstrated in validated protocols

  • Alternatively, 5% BSA in TBST has been used successfully for some antibodies

• Antibody dilution:

  • Primary antibody: Dilute according to manufacturer recommendations (e.g., 1:500-1:2000 for 12347-1-AP or 1:5000-1:50000 for 68293-1-Ig)

  • Secondary antibody: Anti-Rabbit or Anti-Mouse IgG-HRP at 1:1000-1:3000 dilution

• Detection method:

  • Enhanced chemiluminescence (ECL) is suitable for visualizing the 17 kDa MAGOH band

  • Use ImageJ software for signal quantification as demonstrated in melanoma studies

How can researchers effectively distinguish between MAGOH and MAGOHB in experimental settings?

Distinguishing between these highly similar paralogues requires specialized approaches:

• Selective antibodies: While many antibodies recognize both proteins due to high sequence similarity, some are more selective. Verify specificity through dot blot analysis against recombinant MAGOH and MAGOHB proteins .

• RT-qPCR for transcript analysis: Design primers specific to unique regions of MAGOH and MAGOHB mRNAs. Note that at the protein level, MAGOH and MAGOHB cannot be individually assessed by size alone, appearing as a single band in immunoblots that is referred to as MAGOH/MAGOHB .

• Tagged protein expression: Utilize differently tagged versions (e.g., FLAG-MAGOH and V5-MAGOHB) in co-expression studies to examine their competition for binding partners .

• Selective knockdown: Design siRNAs specifically targeting MAGOH or MAGOHB. Studies have used three different siRNAs for each (referred to as 1–3 for MAGOH and 4–6 for MAGOHB) . A non-targeting siRNA pool should be used as control .

• Individual vs. combined knockdown: Compare phenotypes between individual knockdowns and combined MAGOH/MAGOHB depletion to identify unique or redundant functions .

What experimental approaches best elucidate MAGOH's role in cancer progression?

To investigate MAGOH's role in cancer:

• Expression analysis in clinical samples:

  • Compare MAGOH protein levels between tumor and adjacent normal tissues using Western blot and IHC

  • Quantify differences using ImageJ software as demonstrated in LGG studies

  • Analyze public datasets (TCGA, CGGA) for MAGOH mRNA expression across multiple cancer types

• Survival correlation:

  • Stratify patients into high and low MAGOH expression groups

  • Perform Kaplan-Meier survival analysis to correlate expression with patient outcomes

  • Integrate with tumor mutation burden (TMB) data for comprehensive prognostic assessment

• Functional studies in cancer cell lines:

  • Perform MAGOH/MAGOHB knockdown in relevant cell lines (e.g., SW-1088, SW-1783, BT142 for LGG; melanoma cell lines)

  • Assess effects on:

    • Proliferation (cell counting, MTS/MTT assays)

    • Apoptosis (Annexin V staining, caspase activation)

    • Cell cycle progression (flow cytometry)

    • Migration/invasion capabilities

• Mechanistic investigations:

  • Conduct RNA-seq after MAGOH depletion to identify altered splicing events

  • Perform GO-BP and KEGG pathway analyses on differentially expressed genes

  • Analyze changes in immune-related signatures using ssGSEA algorithm

• Genomic correlation studies:

  • Examine relationships between MAGOH expression and:

    • Copy number alterations (CNAs)

    • Somatic mutations (particularly in cancer-related genes like IDH1, CIC, TP53, ATRX)

    • Tumor mutation burden (TMB)

How does MAGOH contribute to immune regulation in the tumor microenvironment?

MAGOH has significant associations with immune features in tumors:

• Immune cell infiltration assessment:

  • Use single-sample GSEA (ssGSEA) algorithm to quantify 29 immune-related factors

  • Apply ESTIMATE algorithm to determine stromal and immune scores, and tumor purity

  • Employ CIBERSORT algorithm to estimate tumor-infiltrating immune cell (TIIC) populations

• Observed immune correlations in LGG:

  • High MAGOH expression correlates with increased immune cell infiltration

  • Positive correlation with infiltration of resting memory CD4+ T cells and activated dendritic cells

  • Negative correlation with M2 macrophages, resting NK cells, and memory B cells

• Checkpoint molecule analysis:

  • Examine correlations between MAGOH expression and immune checkpoint genes (ICPGs)

  • MAGOH expression positively correlates with several ICPGs, including CD28, CD80, CD86, PD1, PD-L1, and CTLA4

• Experimental approaches:

  • Perform co-culture experiments with cancer cells and immune cells after MAGOH manipulation

  • Use flow cytometry to assess changes in immune cell activation markers

  • Evaluate cytokine/chemokine production through ELISA or cytometric bead arrays

  • Conduct in vivo studies using immunocompetent mouse models to assess effects on tumor immunogenicity

What methods are most effective for studying MAGOH's function in the exon junction complex?

To investigate MAGOH's role in the EJC:

• Protein-protein interaction studies:

  • Co-immunoprecipitation of MAGOH/MAGOHB with other EJC components (e.g., EIF4A3, RBM8A)

  • Use tagged versions of MAGOH to pull down interacting partners

  • Employ proximity ligation assays to visualize interactions in situ

• RNA-protein interaction analysis:

  • RNA immunoprecipitation (RIP) to identify RNA targets

  • CLIP-seq (crosslinking immunoprecipitation-sequencing) to map binding sites on RNAs

  • Analyze exon junction associations using computational approaches

• Functional assays for NMD activity:

  • Utilize reporter constructs containing premature termination codons (PTCs)

  • Measure NMD efficiency after MAGOH/MAGOHB knockdown

  • Assess changes in endogenous NMD target transcripts

• Splicing pattern analysis:

  • Perform RNA-seq after MAGOH depletion to identify splicing alterations

  • Focus on exon skipping events, which appear more prevalent in MAGOH/MAGOHB knockdown cells

  • Validate key splicing changes through RT-PCR

• Structural studies:

  • Examine the MAGOH-RBM8A heterodimer and its interaction with EIF4A3

  • Study how this heterodimer regulates EIF4A3 ATPase activity

  • Investigate interactions with PYM1 and the ribosomal 48S pre-initiation complex

How does one resolve contradictory findings regarding MAGOH's effects on apoptosis?

Research shows seemingly contradictory findings about MAGOH's role in apoptosis:

What are the optimal conditions for performing immunohistochemistry with MAGOH antibodies?

For successful IHC with MAGOH antibodies:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissues

  • Validated on human ovary tumor tissue and endometrial adenocarcinoma

• Antigen retrieval:

  • Primary method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • For ab180505: EDTA buffer pH 9 before IHC staining protocol

• Blocking and antibody conditions:

  • Optimal dilutions: 1:50-1:500 for 12347-1-AP; 1:250 for ab180505

  • Secondary detection: HRP Polymer for Rabbit IgG

  • Counterstain: Hematoxylin

• Controls:

  • Positive control: Human ovary tumor tissue or endometrial adenocarcinoma

  • Negative control: Primary antibody omission

  • Perform comparative staining with antibodies to different MAGOH epitopes

• Signal development and visualization:

  • DAB (3,3'-diaminobenzidine) for chromogenic detection

  • Evaluate nuclear and cytoplasmic MAGOH localization

  • Use digital image analysis for quantification when possible

What considerations are important when designing siRNA knockdown experiments for MAGOH/MAGOHB?

For effective MAGOH/MAGOHB knockdown:

• siRNA design strategy:

  • Target both MAGOH and MAGOHB for complete protein depletion

  • Use multiple siRNAs for each gene (e.g., three different siRNAs each for MAGOH and MAGOHB)

  • Include appropriate controls (non-targeting siRNA pools)

• Validation methods:

  • Western blot to confirm protein reduction

  • RT-qPCR to verify mRNA knockdown

  • Note that antibodies typically detect both proteins as a single band

• Experimental design considerations:

  • Test individual knockdowns of MAGOH or MAGOHB

  • Compare with simultaneous knockdown of both

  • Perform time-course experiments to capture both immediate and delayed effects

• Functional readouts:

  • Cell proliferation (can be significantly inhibited by MAGOH knockdown)

  • Apoptosis (induced by MAGOH knockdown, enhanced by simultaneous MAGOHB knockdown)

  • Cell cycle progression (may not be affected by MAGOH knockdown in melanoma)

• Rescue experiments:

  • Express siRNA-resistant MAGOH or MAGOHB constructs

  • Use constructs with silent mutations in the siRNA target sequence

  • Verify whether phenotypes can be reversed by re-expression

What advanced analytical approaches can elucidate MAGOH's impact on alternative splicing events?

To comprehensively analyze MAGOH's effects on splicing:

• RNA-seq experimental design:

  • Compare control vs. MAGOH/MAGOHB knockdown cells

  • Include biological replicates (minimum n=3)

  • Consider time-course experiments to capture dynamic changes

• Splicing pattern analysis:

  • Focus on exon skipping events (two or more exons), which are more prevalent in MAGOH/MAGOHB knockdown cells

  • Use specialized algorithms such as rMATS, MISO, or VAST-TOOLS

  • Calculate percent spliced in (PSI) values for alternative splicing events

• Validation approaches:

  • RT-PCR with primers flanking alternatively spliced regions

  • Minigene reporter assays for key splicing events

  • Targeted RNA-seq for deep coverage of specific genes

• Mechanistic investigations:

  • RNA immunoprecipitation to identify direct MAGOH RNA targets

  • CLIP-seq to map MAGOH binding sites relative to affected splice sites

  • RNA structure analysis to examine how MAGOH binding affects RNA folding

• Integrative analysis:

  • Correlate splicing changes with alterations in gene expression

  • Identify enriched sequence motifs near affected splice sites

  • Perform pathway analysis on genes with altered splicing

How can MAGOH expression be utilized as a biomarker in cancer diagnostics and prognosis?

MAGOH shows promise as a cancer biomarker:

• Prognostic value:

  • High MAGOH expression correlates with adverse prognosis in multiple cancer types

  • Functions as an independent prognostic biomarker in lower-grade glioma (LGG)

  • Integrating MAGOH expression with tumor mutation burden provides enhanced prognostic stratification

• Assessment methodologies:

  • IHC of tumor tissue samples (1:50-1:500 dilution for 12347-1-AP)

  • RT-qPCR for mRNA quantification

  • Analysis of public genomic databases (TCGA, CGGA)

• Biomarker panel integration:

  • Combine MAGOH with other EJC component expression

  • Integrate with established biomarkers for specific cancer types

  • Correlate with immune checkpoint gene expression for immunotherapy response prediction

• Validation approaches:

  • Multi-center cohort studies with standardized assessment protocols

  • Test in different cancer types and stages

  • Compare with current standard of care biomarkers

• Clinical implementation considerations:

  • Standardize cutoff values for high vs. low expression

  • Ensure reproducibility across testing platforms

  • Validate in prospective clinical trials

What is the relationship between MAGOH expression and response to chemotherapy or immunotherapy?

MAGOH shows important associations with therapy responses:

• Chemotherapy correlation:

  • MAGOH expression is associated with responses to chemotherapy in LGG patients

  • Mechanistic studies suggest links to DNA replication pathways

• Immunotherapy implications:

  • MAGOH expression correlates positively with immune checkpoint genes (PD1, PD-L1, CTLA4)

  • High MAGOH expression associates with increased immune cell infiltration

  • These findings suggest potential connections to immunotherapy response

• Experimental approaches:

  • Test chemosensitivity in high vs. low MAGOH-expressing cells

  • Assess immunotherapy response in models with varying MAGOH levels

  • Analyze patient cohorts receiving immunotherapy for correlations with MAGOH expression

• Combination therapy investigations:

  • Study whether MAGOH inhibition could sensitize resistant tumors to therapy

  • Examine synergistic effects with checkpoint inhibitors

  • Explore combinations with targeted therapies based on pathway analysis

• Predictive biomarker development:

  • Develop and validate MAGOH-based scoring systems for therapy response prediction

  • Integrate with other predictive biomarkers (TMB, MSI status, PD-L1 expression)

  • Perform multi-omics analysis for comprehensive response prediction models