MLST8 Antibody

What is MLST8 and what cellular functions does it mediate?

MLST8 (mammalian lethal with sec-13 protein 8) is a 326-amino acid protein with a calculated molecular weight of 36 kDa. It functions as a component of both mTORC1 and mTORC2 complexes and plays a critical role in the regulation of cell growth and survival in response to nutrient and hormone signaling . MLST8 is also known by several aliases including G-protein beta subunit-like (GβL), GBL, Lst8, and Pop3 . The protein is widely expressed in cells, with highest expression observed in skeletal muscle, heart, and kidneys . Structurally, MLST8 consists of seven WD40 repeats that form a β-propeller structure which facilitates protein-protein interactions within the mTOR complexes .

What types of MLST8 antibodies are commonly used in research applications?

Research-grade MLST8 antibodies are available in several formats:

The choice of antibody depends on the specific application and experimental design. Polyclonal antibodies often provide greater sensitivity by recognizing multiple epitopes, while monoclonal antibodies offer higher specificity for particular epitopes .

What is the recommended storage protocol for maintaining MLST8 antibody activity?

For optimal preservation of antibody activity, store MLST8 antibodies at -20°C. Most preparations remain stable for one year after shipment when properly stored. Aliquoting is generally unnecessary for -20°C storage, though smaller volume preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer . The standard storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Repeated freeze-thaw cycles should be avoided to maintain antibody efficacy. For short-term storage, antibodies may be kept at 4°C, though this is not recommended for extended periods .

How should MLST8 antibodies be validated for experimental use?

Proper validation of MLST8 antibodies should include the following steps:

• Positive and negative controls: Use tissue or cell lines known to express MLST8 (e.g., skeletal muscle, kidney) as positive controls. For negative controls, utilize MLST8 knockout cell lines, such as those generated using CRISPR-Cas9 technology .

• Cross-reactivity testing: Confirm species reactivity. Many commercial antibodies show reactivity with human, mouse, and rat MLST8 .

• Specificity verification: For Western blot applications, verify that the antibody detects a band at the expected molecular weight (approximately 36 kDa) .

• Application-specific validation: For immunohistochemistry, validate using paraffin-embedded human kidney tissue at a dilution of 1:100 . For immunofluorescence, use established cell lines such as MCF-7 with appropriate secondary antibodies, such as Alexa Fluor 488-conjugated goat anti-rabbit IgG .

What protocol is recommended for isolating MLST8-protein complexes?

For the isolation of MLST8-containing protein complexes such as mLST8-CCT, the following tandem affinity purification protocol has proven effective:

• Prepare cell lysate at 4°C from cells overexpressing tagged MLST8.

• Load filtered lysate onto a HisTrap HP 5 mL column equilibrated with buffer (20 mM HEPES, 20 mM NaCl, 25 mM imidazole, 0.05% CHAPS, 1 mM TCEP, pH 7.5) .

• Wash column with 5 column volumes of equilibration buffer .

• Apply a linear gradient from 25 to 500 mM imidazole over 8 column volumes .

• Analyze fractions by SDS-PAGE and pool those containing MLST8 and associated proteins .

• For secondary purification, load pooled fractions onto Strep-Tactin resin equilibrated with 20 mM HEPES pH 7.5, 20 mM NaCl .

• Wash twice with buffer containing 150 mM NaCl, then twice with lower salt buffer (20 mM NaCl) .

• Elute complexes with 20 mM HEPES pH 8.0, 20 mM NaCl, 0.05% CHAPS, 2.5 mM d-desthiobiotin .

• Concentrate using a 30 kDa cutoff filter and analyze by SDS-PAGE, Coomassie staining, and immunoblotting .

What are the optimal conditions for using MLST8 antibodies in Western blot applications?

For optimal Western blot results with MLST8 antibodies:

• Sample preparation: Lyse cells in buffer containing protease inhibitors. Centrifuge at high speed (e.g., 15,000×g for 15 minutes at 4°C) to clear debris .

• Protein denaturation: Heat samples at 95°C for 10 minutes in SDS loading buffer .

• Gel selection: Use 10-12% polyacrylamide gels for optimal resolution of the 36 kDa MLST8 protein .

• Transfer conditions: Transfer to PVDF or nitrocellulose membranes using standard protocols.

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST.

• Antibody dilution: For polyclonal antibodies, start with a 1:1000 dilution and optimize as needed. Mouse polyclonal anti-MLST8 antibodies have been successfully used for Western blot applications with similar dilutions .

• Detection: Use appropriate secondary antibodies and chemiluminescent or fluorescent detection methods based on your laboratory's capabilities.

How does MLST8 interact with mTOR complexes and what experimental approaches can identify these interactions?

MLST8 interacts directly with mTOR in both mTORC1 and mTORC2 complexes, enhancing mTOR kinase activity . This interaction is critical for the regulation of protein synthesis, cell growth, and autophagy . To investigate these interactions experimentally:

• Co-immunoprecipitation assays: Overexpress tagged versions of mTOR, raptor, and MLST8 (e.g., AU1-mTOR, HA-raptor, and HA-mLST8) in cells such as 293T. Immunoprecipitate with anti-AU1 antibodies and detect co-precipitated proteins with anti-HA antibodies . This approach can reveal how treatments affect complex formation.

• ATP effects on complex formation: To examine ATP's role in complex stability, incubate immunoprecipitated complexes with ATP (e.g., 5 mM) for varying times (1-3 hours at 4°C) . This helps determine whether ATP affects the association of MLST8 with mTOR complexes.

• Structural analysis: For detailed structural insights, cryo-electron microscopy (cryo-EM) and single-particle 3D reconstruction can be used to characterize MLST8-containing complexes, such as mLST8-CCT intermediates .

What is the role of MLST8 in coronavirus replication and how can this be experimentally investigated?

MLST8 has been identified as a critical host factor for coronavirus replication . Knockout of mLST8 significantly inhibits coronavirus replication through mechanisms involving the mTORC1 signaling pathway and autophagy . To investigate this experimentally:

• Generate mLST8 knockout cells: Use CRISPR-Cas9 technology with specific sgRNAs targeting MLST8 in appropriate cell lines (e.g., PK-15 cells for TGEV studies) .

• Viral infection assays: Infect wild-type and mLST8 KO cells with coronavirus at various MOIs (e.g., 0.01, 0.1, and 1) and assess viral replication by:

  • RT-qPCR quantification of viral RNA

  • Viral titer measurements using plaque assays

  • Immunofluorescence detection of viral proteins (e.g., nucleocapsid protein)

• Rescue experiments: Re-express mLST8 in knockout cells to confirm specificity of the observed effects on viral replication .

• Stage-specific analyses: Determine which stage of the viral life cycle is affected using:

  • Viral adsorption assays (incubation at 4°C for 1 hour)

  • Viral internalization assays (with pronase treatment to remove non-internalized particles)

  • Viral release assays (comparing intracellular and extracellular viral titers)

  • Genome replication assays (using strand-specific RT-PCR to distinguish positive and negative-strand viral RNA)

How does the chaperonin CCT interact with MLST8 and what techniques can visualize this interaction?

The eukaryotic chaperonin CCT plays a key role in mTORC assembly by folding both mLST8 and Raptor . The mLST8-CCT complex can be isolated from cells and visualized using advanced structural biology techniques:

• Isolation of complexes: Tandem affinity purification from cells overexpressing tagged mLST8 can yield purified mLST8-CCT complexes suitable for structural studies .

• Cryo-electron microscopy: High-resolution structural analysis using cryo-EM has revealed that human CCT in complex with mLST8 adopts an open conformation with mLST8 positioned in the center between the two CCT rings .

• Single-particle 3D reconstruction: This technique has achieved 4.0 Å resolution of the mLST8-CCT complex, showing that the apical domains of CCT adopt a very asymmetric arrangement .

• Co-immunoprecipitation with ATP treatment: To investigate the effect of ATP on complex stability, perform co-immunoprecipitation of mLST8 and Raptor with CCT from cells expressing Flag-tagged CCT3 subunit, with varying durations of ATP exposure (e.g., 1-3 hours) .

What factors could affect MLST8 antibody performance and how can these issues be addressed?

Several factors may impact MLST8 antibody performance:

• Epitope accessibility: The WD40 repeat structure of MLST8 may affect epitope accessibility in different experimental conditions. Solution: Try multiple antibodies targeting different epitopes or use denaturing conditions for applications like Western blot .

• Protein-protein interactions: MLST8's interactions with mTOR and other proteins may mask antibody binding sites. Solution: Consider using detergents like CHAPS (0.05%) in buffers to help disrupt protein complexes without denaturing the target protein .

• Post-translational modifications: Modifications may affect antibody recognition. Solution: Use phospho-specific antibodies if investigating particular signaling states .

• Cross-reactivity: Some antibodies may cross-react with related WD40-repeat proteins. Solution: Always validate antibody specificity using knockout controls .

• Fixation sensitivity: For immunohistochemistry and immunofluorescence, fixation conditions can affect epitope recognition. Solution: Optimize fixation protocols (e.g., test both paraformaldehyde and methanol fixation) .

How can researchers distinguish between mTORC1 and mTORC2-associated MLST8 in experimental settings?

Distinguishing between MLST8 in mTORC1 versus mTORC2 complexes requires specific experimental approaches:

• Co-immunoprecipitation with complex-specific components:

  • For mTORC1: Co-IP with antibodies against Raptor, a specific component of mTORC1 .

  • For mTORC2: Co-IP with antibodies against Rictor, a specific component of mTORC2.

• Pharmacological separation:

  • Rapamycin treatment primarily affects mTORC1 but not mTORC2 in acute treatments. Compare MLST8 associations before and after rapamycin treatment .

  • FTS (farnesylthiosalicylic acid) treatment can disrupt the association of raptor with mTOR, affecting mTORC1-associated MLST8. Monitor changes in complex formation at different FTS concentrations (e.g., 30 μM shows half-maximal effect) .

• Downstream signaling analysis:

  • mTORC1 activity: Monitor phosphorylation of 4E-BP1 and S6K1 .

  • mTORC2 activity: Monitor AKT phosphorylation at Ser473 .

  • Changes in these phosphorylation patterns after manipulating MLST8 can indicate which complex is primarily affected.

What approaches can resolve contradictory experimental results when studying MLST8 function?

When facing contradictory results in MLST8 research:

• Cell type considerations: MLST8 function may vary between cell types. Compare results across multiple cell lines relevant to your research question (e.g., HEK293T, MCF-7, PK-15) .

• Genetic manipulation verification: Confirm complete knockout or knockdown by both protein (Western blot) and mRNA (RT-qPCR) analyses .

• Rescue experiments: Re-express MLST8 in knockout cells to determine if the observed phenotypes are specifically due to MLST8 loss .

• Mutational analysis: Create point mutations in key functional regions of MLST8 (e.g., amino acid sites 44, 46, 272, 274) to determine which domains are critical for specific functions .

• Pathway inhibitor controls: Use mTORC1-specific inhibitors (like rapamycin) and mTORC2 inhibitors to dissect which complex mediates particular MLST8 functions .

• Temporal considerations: Some effects may be time-dependent. Conduct time-course experiments (e.g., viral titers at 12, 24, and 36 hours post-infection) to capture the full spectrum of MLST8's effects .

• Complementary techniques: When one technique yields ambiguous results, use complementary approaches. For example, combine biochemical assays with microscopy and functional assays to build a more complete picture .

How can researchers investigate the relationship between STAT3 and MLST8 gene expression?

The relationship between STAT3 and MLST8 gene expression can be investigated using the following approaches:

• ChIP (Chromatin Immunoprecipitation):

  • Use anti-STAT3 antibodies to identify STAT3 binding to the MLST8 promoter region.

  • According to research, the region between -951 to -894 bp of the MLST8 promoter is important for STAT3 binding .

  • Analyze results with qPCR targeting the MLST8 promoter region.

• Promoter mapping:

  • Use luciferase reporter assays with different segments of the MLST8 promoter to identify critical regions for STAT3-mediated transcription .

  • Include STAT3 binding site mutations in these constructs to confirm specificity.

• STAT3 knockdown experiments:

  • Use siRNA to suppress STAT3 expression and monitor effects on:

    • MLST8 mRNA levels (by RT-qPCR)

    • MLST8 protein levels (by Western blot)

    • Downstream signaling effects, such as 4E-BP1 and AKT phosphorylation

• Rescue experiments:

  • In STAT3 knockdown cells, overexpress MLST8 to determine if this restores normal mTORC1/2 signaling, confirming MLST8 as the critical mediator of STAT3's effects on these pathways .

• Functional assays:

  • Assess cap-dependent translation, which is regulated by mTORC1, in cells with STAT3 knockdown with or without MLST8 overexpression .

  • Analyze cell cycle progression, particularly G2/M phase arrest, which can be affected by STAT3 knockdown and partially restored by co-knockdown of 4EBP1 .