MOB3C Antibody

What is MOB3C and what is its primary function in cellular biology?

MOB3C (MOB kinase activator 3C) is a member of the MOB1/phocein protein family. It shares similarity with the yeast Mob1 protein, which binds to Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation . Recent research has uncovered a unique association between MOB3C and the RNase P complex, an endonuclease that catalyzes tRNA 5′ maturation, suggesting MOB3C may play an important role in RNA biology . The protein has a calculated molecular weight of approximately 25.6 kDa, though it may appear at different molecular weights on Western blots depending on post-translational modifications .

For optimal MOB3C detection by Western blot:

• Use freshly prepared cell or tissue lysates with protease inhibitors

• Load 20-50 μg of total protein per lane

• Use appropriate positive control tissues (lung and spleen express MOB3C)

• Start with antibody dilution of 1:1000 in 5% BSA or non-fat milk

• Incubate membrane overnight at 4°C for maximum sensitivity

• Expect to see bands at approximately 22-26 kDa (calculated MW), though some antibodies detect MOB3C at 72 kDa

• Include appropriate loading controls

If you experience non-specific bands, increase blocking time or try different blocking agents to reduce background .

How does MOB3C interact with the RNase P complex, and what methodologies are best for studying this interaction?

MOB3C has been identified to uniquely associate with 7 of 10 protein subunits of the RNase P complex, revealing a novel connection between MOB proteins and RNA biology . To study this interaction:

• BioID proximity labeling: This technique successfully identified the MOB3C-RNase P interaction by expressing BirA*-FLAG-MOB3C fusion proteins to biotinylate proximal proteins .

• Co-immunoprecipitation with crosslinking: Standard co-IP may not identify all interactions, as researchers found that chemical crosslinking with dithiobis(succinimidyl propionate) (DSP) was necessary to capture the MOB3C-RNase P interaction .

• Affinity purification-mass spectrometry (AP-MS): This approach confirmed that MOB3C interacts with assembled RNase P holoenzyme rather than individual subunits .

• Functional validation: Pre-tRNA cleavage assays of MOB3C pulldowns demonstrated that MOB3C associates with catalytically active RNase P complex .

The research by Xiong et al. found that among the seven shared protein subunits between RNase P and MRP complexes (POP1, POP4, RPP14, RPP25, RPP30, RPP38, and RPP40), MOB3C showed strongest association with POP1 but could not be detected in individual POP1 immunoprecipitates, suggesting MOB3C interacts with the assembled holoenzyme .

What are the key considerations for immunohistochemical detection of MOB3C in tissue samples?

For successful immunohistochemical detection of MOB3C:

• Tissue preparation: Use formalin-fixed, paraffin-embedded sections at 4-μm thickness .

• Antigen retrieval: Employ high-pressure and temperature Tris-EDTA (pH 8.0) for optimal antigen retrieval . This step is critical as inadequate retrieval may lead to false-negative results.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide for 30 min, followed by normal goat serum blocking for 30 min to reduce non-specific binding .

• Antibody incubation: Use MOB3C antibody at 1:100-1:300 dilution and incubate overnight at 4°C for best results .

• Detection system: Employ biotin-labeled secondary antibody and horseradish peroxidase-labeled streptavidin working solution, followed by 3,3'-diaminobenzidine chromogenic solution .

• Controls: Always include positive control tissues (pancreas shows good MOB3C expression) and negative controls (antibody pre-absorbed with immunogen peptide) .

• Scoring: For semi-quantitative analysis, calculate immunoreactivity score using both staining intensity and percentage of positive cells .

What is known about differential expression of MOB3C in cancer, and how should researchers approach such studies?

While MOB3C-specific cancer research is limited, studies on the related protein MOB3B provide insights into investigation approaches:

MOB3B expression was reduced in colorectal cancer (CRC) compared to normal tissues, and loss of expression was associated with poor prognosis . By extension, researchers investigating MOB3C in cancer should:

• Tissue comparison: Analyze matched tumor and adjacent normal tissue pairs using IHC and Western blot to assess expression differences .

• Clinical correlation: Examine associations between MOB3C expression and clinicopathological characteristics such as tumor stage, metastasis, and patient survival . A sample correlation table might look like:

• Functional studies: Perform overexpression and knockdown experiments in relevant cell lines to determine effects on proliferation, migration, invasion, and other cancer hallmarks .

• Pathway analysis: Investigate whether MOB3C affects the mTOR/autophagy pathway as observed with MOB3B, or if it functions through RNA processing via RNase P interaction .

• In vivo validation: Confirm in vitro findings using animal models, such as xenograft experiments .

Why might I observe different molecular weights for MOB3C in Western blot experiments?

The calculated molecular weight of MOB3C is approximately 25.6 kDa , but researchers may observe bands at different molecular weights:

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

• Protein isoforms: Alternatively spliced transcript variants of MOB3C encoding distinct isoforms have been observed , which may present at different molecular weights.

• Antibody specificity: Different antibodies target different epitopes, potentially recognizing specific forms of the protein. For example, Boster Bio antibody A30514 notes a calculated MW of 26 kDa but reports observed MW at 72 kDa .

• Sample preparation: Incomplete denaturation or sample buffer issues can affect protein migration.

To address this variability:

• Use multiple antibodies targeting different epitopes to confirm specificity

• Include positive controls with known MOB3C expression

• Perform antibody validation with blocking peptides

• Consider using recombinant MOB3C protein as a reference standard

How do I validate the specificity of MOB3C antibodies?

Rigorous validation of MOB3C antibody specificity is crucial for reliable research results:

• Peptide competition/blocking assay: Pre-incubate the antibody with immunizing peptide before application. This should abolish specific signal, as demonstrated in the immunohistochemical analysis of human pancreas tissue .

• Knockdown/knockout validation: Compare signal between wild-type samples and those with MOB3C knocked down via siRNA/shRNA or knocked out via CRISPR-Cas9.

• Recombinant protein testing: Test against purified recombinant MOB3C protein to confirm recognition.

• Multi-antibody comparison: Use multiple antibodies targeting different epitopes of MOB3C to confirm consistent results.

• Cross-reactivity assessment: Test the antibody against related MOB proteins (MOB1A/B, MOB2, MOB3A/B) to ensure specificity for MOB3C.

• Multiple detection methods: Confirm results across different applications (WB, IHC, IF) when applicable.

• Mass spectrometry validation: For definitive confirmation, perform immunoprecipitation followed by mass spectrometry to verify the identity of the pulled-down protein.

What are the recommended approaches for studying MOB3C proximity interactors?

The BioID proximity labeling technique has proven effective for mapping MOB3C interactors :

• BioID approach: Generate tetracycline-inducible cell lines expressing BirA*-FLAG-MOB3C fusion proteins. Upon activation with biotin, BirA* biotinylates proximal proteins within a ~10 nm radius.

• Controls: Include BirA*-FLAG and BirA*-FLAG-EGFP as negative controls to filter out non-specific interactions .

• Analysis considerations:

  • Apply specificity scoring methods (such as MOB specificity score [MoSS]) to identify true interactors

  • Compare interactomes across different cell lines for robustness

  • Perform functional enrichment analysis to identify biological processes associated with MOB3C interactors

• Validation methods:

  • Co-immunoprecipitation with chemical crosslinking using DSP

  • Affinity purification-mass spectrometry

  • Functional assays (e.g., pre-tRNA cleavage assays for RNase P function)

• Data presentation: Construct protein-protein interaction networks highlighting common and unique interactors compared to other MOB family proteins .

How can I assess the functional role of MOB3C in RNA processing via its RNase P interaction?

Based on the discovery of MOB3C's association with the RNase P complex , researchers can assess its functional role through:

• Pre-tRNA processing assays: Measure the influence of MOB3C on RNase P activity by:

  • Isolating RNase P via MOB3C pulldown and testing activity on pre-tRNA substrates

  • Comparing pre-tRNA processing efficiency in MOB3C-depleted vs. control cells

  • Analyzing accumulation of unprocessed pre-tRNAs after MOB3C knockdown

• RNA-seq analysis: Perform transcriptomic analysis to identify global changes in:

  • tRNA maturation

  • rRNA processing

  • Other non-coding RNA biogenesis

• MOB3C mutational analysis: Create MOB3C variants with mutations in key domains to pinpoint regions important for RNase P interaction and function.

• Cellular localization studies: Determine if MOB3C co-localizes with RNase P in subnuclear structures using immunofluorescence or proximity ligation assays.

• Kinetic measurements: Assess if MOB3C alters the kinetic parameters of RNase P enzymatic activity (Km, Vmax) using purified components and synthetic substrates.

What experimental design considerations are important when comparing different MOB family proteins?

When designing experiments to compare MOB3C with other MOB family proteins:

• Conservation and diversity: Consider sequence similarity between MOB proteins (MOB3A and MOB3B share 82% and 74% sequence identity with MOB3C, respectively) , which may impact antibody cross-reactivity.

• Expression systems: Use consistent expression systems (e.g., same vector backbone, promoter, and tag position) to avoid confounding variables when comparing proteins.

• Subfamily-specific analysis: Group comparisons as follows:

  • MOB1 subfamily (MOB1A, MOB1B): Associated with Hippo pathway

  • MOB2: Interacts with NDR kinases

  • MOB3 subfamily (MOB3A, MOB3B, MOB3C): Less characterized, with MOB3C uniquely associated with RNase P

  • MOB4: STRIPAK complex component

• Interactome comparisons: When performing interactome studies, apply:

  • Network analysis to identify common and differential interactors

  • Hierarchical clustering of proximity profiles

  • GO term enrichment to identify functional differences

• Control for expression levels: Ensure comparable expression levels of different MOB proteins to make valid comparisons of interaction strength or functional effects.

• Cell type considerations: Different MOB proteins may have cell type-specific functions, so perform experiments in multiple cell lines for comprehensive comparison .

How should I quantify and normalize MOB3C protein levels in comparative studies?

For accurate quantification of MOB3C protein expression:

• Western blot quantification:

  • Use digital imaging systems with linear dynamic range

  • Perform densitometry analysis with software like ImageJ

  • Run a dilution series to ensure measurements are within linear range

  • Normalize to appropriate loading controls (GAPDH, β-actin, or α-tubulin)

• IHC quantification:

  • Apply semi-quantitative scoring systems that account for both staining intensity and percentage of positive cells

  • The recommended formula: Score = staining intensity × percentage of positive cells

  • Use automated image analysis software for unbiased assessment

  • Include multiple fields per sample (minimum 5)

• Statistical considerations:

  • Use paired tests for matched tumor/normal samples

  • Apply non-parametric tests for IHC scoring data (Mann-Whitney or Kruskal-Wallis)

  • Perform correlation analysis between MOB3C expression and clinical parameters using appropriate tests (Chi-square, Spearman's rank)

• Data presentation:

  • Present individual data points along with means/medians

  • Use box plots for IHC data to show distribution

  • Include representative images for different expression categories

What are the most appropriate experimental models for studying MOB3C function?

Selection of appropriate experimental models for MOB3C research depends on the specific research question:

• Cell line selection:

  • HEK293 and HeLa cells have been successfully used for MOB3C proximity labeling studies

  • RAW264.7 macrophage cells show detectable levels of endogenous MOB3C

  • Consider tissue-specific cell lines based on MOB3C expression profiles (lung and spleen show higher expression)

• Animal models:

  • Mouse models are appropriate as MOB3C is conserved between human and mouse

  • Consider conditional knockout models for tissue-specific studies

  • For cancer studies, xenograft models with MOB3C-manipulated cell lines have been informative for related MOB proteins

• Expression systems:

  • Tetracycline-inducible systems allow controlled expression for interactome studies

  • N-terminal tagging strategies have proven successful for MOB family proteins

  • Consider both overexpression and knockdown/knockout approaches for comprehensive functional analysis

• Primary cell cultures:

  • May provide more physiologically relevant context than immortalized cell lines

  • Validate MOB3C antibody in primary cells before extensive studies

• 3D culture systems:

  • Consider organoid models for studying MOB3C in a tissue-like context

  • Particularly relevant if investigating potential roles in cell polarity or tissue organization