MAST1 Antibody

What is MAST1 and what are its key structural domains?

MAST1 belongs to the family of microtubule-associated serine/threonine kinases, characterized by a unique structural feature combining a serine/threonine kinase domain and a postsynaptic density protein-95/discs large/zona occludens-1 (PDZ) domain. The protein has a calculated molecular weight of approximately 171 kDa (1570 amino acids) . MAST1 contains a domain of unknown function (DUF1908) that includes a four-helix bundle, which is a site for several disease-associated mutations . MAST1 associates with microtubules in a MAP-dependent manner, making it important for cytoskeletal function .

How does MAST1 interact with the microtubule cytoskeleton?

MAST1 binds to microtubules through microtubule-associated proteins (MAPs) rather than directly. In vitro transcription and translation experiments using radiolabeled murine Mast1 (which shares 94% sequence identity with human MAST1) have demonstrated that MAST1 associates with Taxol-stabilized microtubules in a MAP-dependent manner. Mutations in MAST1, particularly the K276 deletion, can significantly enhance MAST1 binding to microtubules, suggesting that alterations in MAST1-microtubule interactions may contribute to pathological processes .

What is the cellular and tissue expression pattern of MAST1?

MAST1 is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. Expression analysis in mouse brain shows that Mast1 expression begins at embryonic day 12.5 (E12.5), peaks at E16.5, and decreases postnatally . In human fetal brain, MAST1 shows moderate expression at gestation weeks 13 and 22. In mature neurons, MAST1 staining has a punctate appearance, suggesting association with vesicular structures trafficked along the microtubule cytoskeleton. Adult mouse expression analysis indicates persistent MAST1 expression in all brain regions, albeit at lower levels, and presence in other tissues including testes .

What criteria should be considered when selecting a MAST1 antibody for research?

When selecting a MAST1 antibody, researchers should consider:

• Target specificity: Verify the antibody has been validated against both positive controls (tissues/cells known to express MAST1) and negative controls (MAST1 knockout samples)

• Applications validated: Ensure the antibody has been tested for your specific application (WB, IP, IF, IHC)

• Species reactivity: Confirm reactivity with your experimental species (human, mouse, rat)

• Epitope location: Consider whether N-terminal, C-terminal, or domain-specific antibodies best suit your research question

• Antibody type: Polyclonal antibodies offer higher sensitivity but potentially lower specificity compared to monoclonals

For MAST1 research, available antibodies have been validated in applications including Western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence, with confirmed reactivity against human, mouse, and rat samples .

How can I validate the specificity of a MAST1 antibody?

A comprehensive validation approach for MAST1 antibodies should include:

• Knockout/knockdown controls: Use MAST1 knockout tissues/cells or MAST1 knockdown via RNAi as negative controls. Several studies have validated MAST1 antibodies using this approach in cultured P0 cortical neurons and P7 cerebellar granule neurons .

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

• Detection of expected molecular weight: MAST1 should appear at approximately 171 kDa in Western blot applications .

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of MAST1 to confirm findings.

• Correlation with mRNA expression: Compare protein detection with RT-PCR or RNA-seq data showing MAST1 expression patterns.

How does MAST1 contribute to neuronal development?

MAST1 plays a crucial role in neuronal differentiation and cell cycle regulation during neurodevelopment:

• Expression pattern: MAST1 is upregulated during neuronal differentiation, with expression increasing from embryonic day 12.5, peaking at E16.5, and decreasing postnatally in mice .

• Neuronal differentiation: Inhibition of MAST1 expression by RNA interference attenuates neuronal differentiation of the human neuroblastoma cell line SH-SY5Y. MAST1 knockdown results in reduced expression of neuronal markers like MAP2 and impaired neurite extension .

• Cell cycle regulation: MAST1 is involved in cell cycle exit during neuronal differentiation. MAST1-depleted cells fail to undergo appropriate cell cycle arrest during differentiation, showing increased EdU-positive cells .

• P27 pathway: MAST1 influences neuronal differentiation through regulation of P27, a cyclin-dependent kinase inhibitor. P27 levels increase during neuronal differentiation, and MAST1 knockdown reduces P27 expression. Importantly, P27 overexpression can partially rescue the reduced neuronal differentiation caused by MAST1 depletion .

What is the role of MAST1 mutations in neurodevelopmental disorders?

MAST1 mutations have been implicated in several neurodevelopmental disorders:

• Mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM): De novo mutations in MAST1 cause this syndrome in the absence of megalencephaly. Three of the identified variants in MCC-CH-CM patients were single amino acid deletions positioned in the hydrophobic core of a four-helix bundle in the DUF1908 domain (p.Glu194del, p.Lys276del, p.Leu278del), while others harbored a recurrent missense mutation in the kinase domain (p.Gly517Ser) .

• Other neurodevelopmental phenotypes: De novo MAST1 mutations have been identified in patients presenting with either microcephaly accompanied by motor deficits or autism spectrum disorder .

• Intellectual disability: A novel missense variant c.3539T>G, p.(Leu1180Arg) in MAST1 was identified in a patient with intellectual disability, microcephaly, and dysmorphic features. This and other reported missense variants are predicted to be damaging through in silico pathogenicity prediction analyses .

• Dominant negative mechanism: Animal studies suggest that MAST1 mutations act through a dominant negative mechanism, as Mast1 null animals are phenotypically normal, whereas animals with specific microdeletions (e.g., L278del) recapitulate neurological phenotypes observed in patients .

How do I design experiments to study MAST1's role in neuronal differentiation?

To investigate MAST1's function in neuronal differentiation:

• Cell model selection:

  • SH-SY5Y neuroblastoma cells: These cells can be differentiated into neuron-like cells using retinoic acid (RA) treatment, providing a well-established model to study MAST1's role in neuronal differentiation

  • Primary neuron cultures: Cortical neurons or cerebellar granule neurons from P0-P7 mice or rats offer a more physiologically relevant system

• Experimental approaches:

  • MAST1 knockdown: Use shRNA or siRNA targeting MAST1 to reduce expression, then assess effects on neuronal differentiation markers (MAP2, TUBB3) and morphology

  • MAST1 overexpression: Express wild-type or mutant MAST1 to study gain-of-function or dominant-negative effects

  • Rescue experiments: After MAST1 knockdown, attempt rescue with expression of downstream effectors like P27 to confirm pathway relationships

• Assessment methods:

  • Immunofluorescence for neuronal markers (MAP2, TUBB3)

  • Morphological analysis of neurite extension

  • Cell cycle analysis using EdU incorporation or flow cytometry

  • Western blot for MAST1 and differentiation markers

  • qPCR for gene expression changes

• Timeline for differentiation studies:
  Based on published protocols, monitor MAST1 expression in SH-SY5Y cells from day 1 through day 8 of RA-induced differentiation, with peak expression typically observed around day 5 .

How does MAST1 contribute to cisplatin resistance in cancer?

MAST1 has emerged as a critical driver of cisplatin resistance in human cancers through specific mechanisms:

• MAPK pathway rewiring: Cisplatin treatment inhibits the MAPK pathway by dissociating cRaf from MEK1. MAST1 can replace cRaf to reactivate the MAPK pathway in a cRaf-independent manner, thereby promoting cancer cell survival .

• Synthetic lethality: MAST1 was identified through a kinome RNAi screen as a synthetic lethal partner with cisplatin. Knockdown of MAST1 sensitizes cancer cells specifically to cisplatin but not to other DNA-damaging agents or chemotherapeutics like paclitaxel .

• Expression correlation: MAST1 expression levels (both protein and mRNA) are upregulated in cisplatin-resistant cells that have been chronically exposed to cisplatin, compared to parental cells. Clinical evidence shows that both initial and cisplatin-induced expression of MAST1 contributes to platinum resistance and worse clinical outcomes .

• Mechanism specificity: MAST1 contributes to cisplatin resistance not by altering DNA damage response or repair pathways, but by activating MEK-mediated anti-apoptotic signaling. MAST1 knockdown does not impact the Fanconi anemia DNA repair pathway, DNA damage response signaling, or cisplatin adduct accumulation/removal .

What experimental methods can be used to study MAST1's role in cancer drug resistance?

To investigate MAST1 in cancer drug resistance:

• Cell models:

  • Paired cisplatin-sensitive and cisplatin-resistant cancer cell lines (KB-3-1, A549, HeLa)

  • Patient-derived xenograft (PDX) models

• Gene manipulation approaches:

  • MAST1 knockdown: shRNA targeting MAST1 to assess effects on cisplatin sensitivity

  • MAST1 overexpression: Wild-type vs. kinase-dead (DA) MAST1 to determine if kinase activity is required

• Analytical methods:

  • Cell viability assays: MTT or similar assays with cisplatin treatment in MAST1-manipulated cells

  • Western blot: Assess MAST1 and downstream MAPK pathway components (MEK1/2 phosphorylation)

  • In vivo tumor growth: Xenograft models with MAST1 knockdown/inhibition plus cisplatin treatment

  • Proximity ligation assay (PLA): To detect protein-protein interactions involving MAST1

  • MAST1 kinase activity assays: ADP-Glo MAST1 kinase assay using inactive recombinant MEK1 as a substrate

• Small molecule inhibitors:

  • Lestaurtinib has been identified as a MAST1 inhibitor that can restore cisplatin sensitivity

  • 17-AAG (Hsp90 inhibitor) decreases MAST1 protein levels and can be used in combination with lestaurtinib for enhanced effects

How is MAST1 protein stability regulated in cancer cells?

MAST1 protein stability in cancer cells is regulated through several mechanisms:

• Hsp90 chaperone interaction: Hsp90B was identified as a binding partner of MAST1 through 2D gel electrophoresis-based proteomic profiling. Endogenous interaction between Hsp90B and MAST1 has been demonstrated in cisplatin-resistant sublines of human carcinoma KB-3-1 and lung cancer A549 cells .

• Isoform specificity: While both Hsp90 isoforms (Hsp90A and Hsp90B) bind to MAST1, Hsp90B is the predominant binding partner. Treatment with the Hsp90 inhibitor 17-AAG diminishes MAST1 protein levels in a dose-dependent manner, while MAST1 mRNA expression remains unchanged, indicating post-transcriptional regulation .

• Deubiquitination: USP1 has been identified as a deubiquitinase that interacts with, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. Expression analysis across human clinical tissues reveals a positive correlation between USP1 and MAST1 .

• Therapeutic implications: The regulatory mechanisms of MAST1 protein stability provide additional therapeutic targets. Combinatorial treatment with the Hsp90 inhibitor 17-AAG and the MAST1 inhibitor lestaurtinib synergistically decreases cell viability of diverse cisplatin-resistant cancer cell lines and further sensitizes cells to cisplatin treatment .

What are the optimal conditions for Western blot detection of MAST1?

For optimal Western blot detection of MAST1:

• Sample preparation:

  • For brain tissue: Homogenize in RIPA buffer with protease inhibitors

  • For cell lines (SH-SY5Y, KB-3-1, A549): Lyse in RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

• Gel electrophoresis parameters:

  • Use 6-8% SDS-PAGE gels due to MAST1's large size (171 kDa)

  • Run at lower voltage (80-100V) for better resolution of high molecular weight proteins

  • Include molecular weight markers that extend to 200 kDa

• Transfer conditions:

  • For proteins >150 kDa, use wet transfer at low voltage (30V) overnight at 4°C

  • Consider adding 0.1% SDS to transfer buffer to improve transfer of large proteins

• Antibody conditions:

  • Primary antibody dilution: 1:500-1:2000 (optimize for specific antibody)

  • Incubation: Overnight at 4°C

  • Secondary antibody: Anti-rabbit HRP (as most MAST1 antibodies are rabbit polyclonals)

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate optimized for high molecular weight proteins

  • Expected band size: 171 kDa

• Controls:

  • Positive control: Brain tissue lysates (mouse/rat/human)

  • Negative control: MAST1 knockdown or knockout samples

What methods can be used to study MAST1 protein-protein interactions?

Several complementary methods can be employed to study MAST1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • MAST1 antibodies have been successfully used for IP with 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Both endogenous and tagged (GST, FLAG) approaches have been validated

  • Example: Endogenous interaction between Hsp90B and MAST1 was demonstrated by Co-IP in cisplatin-resistant cancer cells

• Proximity Ligation Assay (PLA):

  • The Duolink in situ PLA kit has been used to visualize interactions between USP1 and MAST1

  • This technique allows visualization of protein interactions in situ with subcellular resolution

• GST pulldown assays:

  • GST-fused MAST1 has been used to identify binding partners through pulldown experiments followed by mass spectrometry

  • This approach identified Hsp90B as a MAST1 interactor

• Microtubule binding assays:

  • In vitro transcription and translation (TnT) reactions with radiolabeled MAST1 followed by assessing binding to Taxol-stabilized microtubules in the presence or absence of MAPs

  • This method demonstrated MAST1's MAP-dependent association with microtubules

• Yeast two-hybrid screening:

  • While not explicitly mentioned in the search results, this is a complementary approach to identify novel MAST1 interactors

• Mass spectrometry-based approaches:

  • 2D gel electrophoresis followed by MS analysis has successfully identified MAST1 interactors

How can I visualize MAST1 subcellular localization in different cell types?

To visualize MAST1 subcellular localization:

• Immunofluorescence protocol for fixed cells:

  • Fix cells with 4% paraformaldehyde (15 min)

  • Permeabilize with 0.1% Triton X-100 (5 min)

  • Block with 5% bovine serum albumin

  • Incubate with MAST1 primary antibody overnight at 4°C

  • Wash and incubate with appropriate Alexa Fluor-conjugated secondary antibodies (1 hour)

  • Counterstain with DAPI for nuclear visualization

  • Mount using VectaShield or similar medium

• Co-staining strategies:

  • For neuronal compartment analysis: Co-stain with the axonal marker Tau or the dendritic marker Map2

  • For microtubule association: Co-stain with α-tubulin or β-tubulin

  • For subcellular compartments: Use markers for ER (Calnexin), Golgi (GM130), or other relevant organelles

• Cell types validated for MAST1 immunostaining:

  • Neuronal cells: Primary cortical neurons, cerebellar granule neurons, SH-SY5Y neuroblastoma cells

  • Cancer cells: KB-3-1, A549, HeLa cell lines

• Imaging considerations:

  • Use confocal microscopy for precise subcellular localization

  • In neurons, MAST1 shows punctate staining throughout the soma, as well as in dendritic and axonal compartments

  • For mutant MAST1 localization studies, wild-type MAST1 and disease-associated variants appear to localize in similar patterns, suggesting disease mechanisms may not involve mis-targeting

Why might I be getting non-specific bands in my MAST1 Western blot?

Non-specific bands in MAST1 Western blots can occur for several reasons:

• Antibody-specific issues:

  • Cross-reactivity with other MAST family members (MAST2, MAST3, MAST4): These proteins share sequence homology with MAST1, particularly in the kinase and PDZ domains

  • Batch variation: Different lots of the same antibody may show variable specificity

  • Species cross-reactivity: Antibodies raised against human MAST1 may show different specificity patterns in mouse or rat samples

• Technical considerations:

  • Insufficient blocking: Increase blocking time or change blocking agent (BSA vs. milk)

  • Antibody concentration: Try more dilute antibody solutions

  • Sample degradation: Fresh preparation of samples with complete protease inhibitors can reduce degradation products

  • Wash stringency: Increase wash times or add more detergent (0.1% Tween-20) to reduce non-specific binding

• Validation approaches:

  • MAST1 knockout/knockdown controls: MAST1-specific bands should disappear or be reduced

  • Pre-absorption with immunizing peptide: This should eliminate specific bands

  • Comparison across multiple MAST1 antibodies targeting different epitopes: True MAST1 bands should be consistent

• Expected MAST1 band: The full-length protein should appear at approximately 171 kDa .

How can I address weak or absent MAST1 signal in immunostaining experiments?

When facing weak or absent MAST1 immunostaining signals:

• Antigen retrieval optimization:

  • For FFPE tissues: Test different antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • For cell cultures: Optimize fixation conditions (4% PFA for 15 min has been validated)

  • Test different permeabilization methods (0.1% Triton X-100, 0.1% Saponin, or methanol)

• Signal amplification strategies:

  • Tyramide signal amplification (TSA)

  • Biotin-streptavidin amplification systems

  • Use of more sensitive detection systems (Super-Resolution microscopy)

• Expression considerations:

  • Timing: MAST1 expression is developmentally regulated, peaking at E16.5 in mice and decreasing postnatally

  • Cell type specificity: MAST1 is predominantly expressed in post-mitotic neurons

  • Expression level: Consider that baseline expression might be low in some tissues/cells

• Antibody selection:

  • Try multiple antibodies targeting different epitopes

  • For low abundance proteins, monoclonal antibodies may provide better signal-to-noise ratio

• Technical considerations:

  • Fresh antibody dilutions

  • Extended primary antibody incubation (overnight at 4°C is recommended)

  • Reduced washing stringency

  • Use of background reducing agents in antibody diluent

How do I distinguish between MAST1 and other MAST family members in my experiments?

Distinguishing between MAST family members (MAST1-4) requires careful experimental design:

• Antibody selection strategies:

  • Choose antibodies targeting unique regions rather than conserved domains

  • Target N-terminal or C-terminal regions, which typically have lower sequence homology than kinase or PDZ domains

  • Validate antibody specificity against recombinant MAST family proteins

• Gene expression analysis:

  • Design qPCR primers targeting unique regions of each MAST gene

  • Use transcript-specific probes for in situ hybridization

  • RNA-seq data can distinguish between transcripts based on unique sequence reads

• Genetic approaches:

  • Gene-specific knockdown: Use siRNAs or shRNAs targeting unique regions of MAST1

  • CRISPR/Cas9 knockout: Generate specific MAST1 knockout lines as definitive controls

  • Rescue experiments: The specificity of MAST1 knockdown phenotypes can be confirmed by rescue with MAST1 but not other MAST family members

• Functional distinction:

  • MAST1 mutations show distinct phenotypes: While Mast1 null animals are phenotypically normal, specific microdeletions (e.g., L278del) produce neurological phenotypes

  • Different roles in signaling: For example, in cisplatin-resistant cancer cells, MAST1 microdeletions leave the PI3K/AKT3/mTOR pathway unperturbed but diminish Mast2 and Mast3 levels

What emerging technologies might advance MAST1 research?

Emerging technologies with potential to advance MAST1 research include:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM, STED) to visualize MAST1's punctate distribution and interaction with microtubules at nanoscale resolution

  • Live-cell imaging with tagged MAST1 to track dynamic interactions and trafficking

  • Lattice light-sheet microscopy for long-term, low-phototoxicity imaging of MAST1 dynamics

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) to identify the MAST1 interactome in specific subcellular compartments

  • Phosphoproteomics to identify MAST1 substrates and signaling networks

  • Thermal proteome profiling to discover compounds binding to MAST1

• Structural biology techniques:

  • Cryo-EM to determine the structure of MAST1 alone and in complex with binding partners

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study conformational dynamics

  • AlphaFold or similar AI-based structural prediction to model MAST1 domains and mutations

• Gene editing technologies:

  • CRISPR base editing or prime editing to introduce specific MAST1 mutations

  • CRISPR activation/interference systems to modulate MAST1 expression

  • CRISPR screens to identify synthetic lethal interactions with MAST1 in cancer contexts

• Single-cell approaches:

  • Single-cell RNA-seq to characterize MAST1 expression across cell types

  • Single-cell proteomics to analyze MAST1 protein levels and modifications

  • Spatial transcriptomics to map MAST1 expression in tissue contexts

What are the most promising therapeutic avenues targeting MAST1?

Promising therapeutic approaches targeting MAST1 include:

• MAST1 kinase inhibitors:

  • Lestaurtinib has been identified as a MAST1 inhibitor that restores cisplatin sensitivity in cancer cells

  • Further development of selective MAST1 inhibitors could provide more targeted therapies with fewer off-target effects

• Combinatorial approaches:

  • MAST1 inhibition (lestaurtinib) combined with Hsp90 inhibition (17-AAG) has shown synergistic effects in overcoming cisplatin resistance in cancer cells and PDX models

  • Combined targeting of USP1 (regulating MAST1 stability) and MAST1 has sensitized tumors to cisplatin treatment in mouse xenograft models

• Targeting MAST1 protein stability:

  • Disrupting interactions with Hsp90B, which stabilizes MAST1 protein

  • Inhibiting USP1, a deubiquitinase that prevents MAST1 degradation

• Pathway-based approaches:

  • Targeting MAST1-specific activation of the MEK pathway in cisplatin-resistant cancers

  • Exploring the relationship between MAST1 and P27 for neurological disorders

• Precision medicine applications:

  • MAST1 expression as a biomarker for cisplatin response prediction

  • Mutation-specific approaches for neurodevelopmental disorders caused by different MAST1 mutations

What are the key unanswered questions in MAST1 biology?

Several critical questions remain to be addressed in MAST1 research:

• Structural and functional questions:

  • What is the three-dimensional structure of full-length MAST1?

  • What are the physiological substrates of MAST1 kinase activity?

  • How is MAST1 activity regulated in different cellular contexts?

  • What is the function of the DUF1908 domain where several disease-causing mutations are located?

• Developmental neurobiology:

  • How does MAST1 coordinate neuronal differentiation and cell cycle exit during development?

  • What is the mechanism by which MAST1 regulates P27 expression?

  • Why do Mast1 null animals lack phenotypes while specific point mutations cause severe neurodevelopmental disorders?

  • How do different MAST1 mutations lead to diverse neurological phenotypes?

• Cancer biology:

  • Why does MAST1 specifically affect response to cisplatin but not other DNA-damaging agents?

  • How does MAST1 recognize and replace cRaf to activate MEK1?

  • What regulates MAST1 upregulation in response to cisplatin treatment?

  • Can MAST1 targeting strategies overcome other treatment resistances beyond cisplatin?

• Therapeutic development:

  • How can MAST1 inhibitors be optimized for better specificity and pharmacokinetics?

  • What patient populations would benefit most from MAST1-targeted therapies?

  • What biomarkers can predict response to MAST1-targeted treatments?

  • Are there context-dependent advantages to activating versus inhibiting MAST1?