FAM118B Antibody

What is FAM118B and what are its primary cellular functions?

FAM118B (Family with sequence similarity 118, member B) is a nuclear protein with a molecular weight of approximately 38-39 kDa that functions as a critical component of Cajal bodies. Cajal bodies are specialized and dynamic compartments in the nucleus involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs) . FAM118B plays an essential role in maintaining the canonical morphology of Cajal bodies and supporting pre-mRNA splicing processes within the cell.

Research indicates that FAM118B is widely expressed across multiple cell types derived from various tissue origins, suggesting it serves a fundamental cellular function . The protein contains a conserved SIR2-like domain between residues 159 and 301, which may contribute to its functional activities in nuclear organization . FAM118B's most significant role appears to be in regulating symmetric dimethylarginine modification of SmD1 protein, which is crucial for snRNP biogenesis and ultimately affects splicing efficiency .

How does FAM118B interact with other components of Cajal bodies?

FAM118B demonstrates specific interactions with core Cajal body components, particularly coilin (the marker protein of Cajal bodies) and the survival of motor neuron (SMN) protein complex. Co-immunoprecipitation experiments have confirmed that both coilin and SMN can be detected in immunoprecipitates of endogenous FAM118B . Interestingly, while FAM118B shows strong interaction with SMN in reciprocal immunoprecipitation experiments, the interaction between FAM118B and coilin appears to be more nuanced.

In vitro pull-down assays using bacterially expressed MBP-tagged FAM118B and GST-tagged coilin have demonstrated that FAM118B can directly bind to coilin . This direct interaction is functionally relevant, as FAM118B localization to Cajal bodies is dependent on coilin. When coilin is depleted using siRNA, FAM118B loses its concentrated localization in Cajal bodies and becomes diffusely distributed throughout the nucleoplasm . These findings suggest that coilin serves as a scaffold for FAM118B recruitment to Cajal bodies.

What happens to cellular function when FAM118B is depleted?

Depletion of FAM118B through siRNA-mediated knockdown leads to several significant cellular phenotypes that highlight its importance in nuclear organization and RNA processing:

What molecular mechanisms underlie FAM118B's role in pre-mRNA splicing?

FAM118B contributes to pre-mRNA splicing through multiple interconnected molecular mechanisms centered on snRNP biogenesis and modification. The protein's effect on splicing appears to be mediated primarily through its regulation of symmetric dimethylarginine (sDMA) modification of SmD1 protein.

Research shows that FAM118B depletion reduces SmD1 sDMA modification, which in turn diminishes the binding of SMN to this Sm protein . This disruption in SMN-Sm protein interaction impairs snRNP assembly, as the SMN complex is responsible for loading Sm proteins onto snRNAs to form functional snRNPs. Consequently, cells depleted of FAM118B show significant reductions in splicing efficiency.

Quantitative real-time PCR analysis of eight endogenous genes (DPP8, DDX20, NOSIP, ACP1, CSDA, CD44, STK6, and ZNF207) has demonstrated that FAM118B depletion reduces the levels of spliced mRNA by 10-63% . This range of effects suggests that FAM118B may impact different pre-mRNA splicing events to varying degrees, possibly depending on the specific splicing components required for each transcript.

Importantly, the splicing defects observed in FAM118B-depleted cells can be rescued by reintroducing wild-type FAM118B, confirming that the observed phenotypes are directly attributable to FAM118B function rather than off-target effects .

Which domains of FAM118B are critical for its localization to Cajal bodies?

FAM118B contains distinct functional domains that contribute differently to its subcellular localization and interaction with Cajal body components. Truncation mutant analysis has identified specific regions required for targeting FAM118B to Cajal bodies:

Based on this truncation analysis, the region spanning residues 114-232 appears to be essential for targeting FAM118B to Cajal bodies . Interestingly, this region partially overlaps with the predicted SIR2-like domain (residues 159-301), suggesting that this enzymatic domain may contribute to the protein's proper localization.

The middle portion of FAM118B (residues 114-232) is sufficient for Cajal body localization, while neither the N-terminal (residues 1-113) nor the C-terminal (residues 233-351) regions alone can direct the protein to Cajal bodies . These findings indicate that specific sequence elements within the middle portion of FAM118B mediate its interaction with coilin and/or other Cajal body components.

How does FAM118B regulate post-translational modifications of Sm proteins?

FAM118B plays a crucial role in regulating the symmetric dimethylarginine (sDMA) modification of SmD1, a core component of snRNPs. This post-translational modification is essential for the proper interaction between Sm proteins and the SMN complex during snRNP assembly.

Research has demonstrated that depletion of FAM118B results in a significant reduction in the sDMA modification of SmD1 . This modification, which occurs on arginine residues, is typically catalyzed by protein arginine methyltransferases (PRMTs). Although the exact molecular mechanism by which FAM118B promotes this modification remains to be fully elucidated, the presence of a SIR2-like domain in FAM118B suggests it may have enzymatic activity that directly or indirectly influences the methylation state of SmD1.

What are the optimal protocols for detecting FAM118B via Western blotting?

For effective detection of FAM118B via Western blotting, researchers should consider the following optimized protocol based on validated antibodies:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration using a Bradford or BCA assay

  • Load 20-50 μg of total protein per lane

• Electrophoresis and transfer:

  • Separate proteins on a 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary anti-FAM118B antibody at a dilution of 1:500-1:3000 in blocking buffer overnight at 4°C

  • For optimal results with the Prestige Antibodies® system, use a concentration of 0.04-0.4 μg/mL

  • Wash membrane 3 times with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

  • Wash membrane 3 times with TBST, 5 minutes each

• Detection:

  • Apply ECL substrate and expose to X-ray film or image using a digital imager

  • Expected band size for FAM118B is approximately 38-39 kDa

• Validation controls:

  • Include a positive control (HeLa cells or human brain tissue lysate)

  • For negative control, include samples treated with FAM118B siRNA to confirm antibody specificity

This protocol has been successfully used to detect endogenous FAM118B in various cancer cell lines from different tissue origins . The antibody recognizes a major band at about 34 kDa, which corresponds to the predicted molecular mass of endogenous FAM118B .

How should immunohistochemistry experiments with FAM118B antibodies be designed?

Designing effective immunohistochemistry (IHC) experiments for FAM118B detection requires careful consideration of tissue preparation, antigen retrieval, and antibody concentration:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm thickness)

  • Human brain tissue has been validated as a positive control for FAM118B IHC

• Antigen retrieval:

  • Perform heat-induced epitope retrieval (HIER)

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: citrate buffer at pH 6.0

  • Heat in a pressure cooker or microwave until boiling, then maintain at sub-boiling temperature for 10-20 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum (from the same species as the secondary antibody) for 1 hour

  • Apply primary anti-FAM118B antibody at a dilution of 1:20-1:200

  • For Prestige Antibodies® system, use a dilution range of 1:500-1:1000

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3 times with PBS, 5 minutes each

  • Apply appropriate biotinylated secondary antibody for 30 minutes at room temperature

  • Wash 3 times with PBS, 5 minutes each

• Detection and visualization:

  • Apply avidin-biotin complex (ABC) or polymer-based detection system

  • Develop with DAB (3,3'-diaminobenzidine) substrate

  • Counterstain with hematoxylin, dehydrate, clear, and mount

• Subcellular localization:

  • FAM118B should primarily show nuclear localization

  • In cells with intact Cajal bodies, expect to see concentrated foci within the nucleus

  • Following Triton X-100 extraction of cytosolic and nucleoplasmic proteins, the colocalization of FAM118B with coilin in Cajal bodies becomes more pronounced

• Controls:

  • Positive control: Include human brain tissue sections, which have been validated for FAM118B expression

  • Negative control: Omit primary antibody on duplicate sections

  • Technical validation: Consider using tissues from FAM118B-knockdown models if available

What controls should be included when validating FAM118B antibody specificity?

Validating the specificity of FAM118B antibodies is crucial for ensuring reliable experimental results. Comprehensive validation should include multiple complementary approaches:

• RNA interference control:

  • Treat cells with siRNA targeting FAM118B

  • The 34 kDa band corresponding to FAM118B should be significantly reduced in Western blots of lysates from these cells

  • This approach has been successfully used to validate FAM118B antibody specificity, showing that the band is greatly reduced after siRNA-mediated depletion

• Overexpression control:

  • Transfect cells with a plasmid expressing tagged FAM118B (e.g., HA-FLAG-tagged)

  • Confirm that the antibody detects both endogenous FAM118B and the overexpressed tagged version

  • The tagged version will appear at a slightly higher molecular weight due to the additional tag

• Peptide competition assay:

  • Pre-incubate the FAM118B antibody with the immunizing peptide before application

  • This should block specific binding and eliminate the FAM118B signal

  • A control incubation with an unrelated peptide should not affect antibody binding

• Cross-reactivity assessment:

  • Test the antibody against lysates from different species to confirm the expected cross-reactivity pattern

  • Commercial FAM118B antibodies have been validated to react with human, mouse, and rat samples

• Immunoprecipitation validation:

  • Perform immunoprecipitation with the FAM118B antibody

  • Analyze the precipitate by mass spectrometry to confirm the presence of FAM118B

  • Western blot analysis of the immunoprecipitate should show enrichment of FAM118B

• Immunofluorescence colocalization:

  • Perform dual immunofluorescence staining with two different antibodies against FAM118B (targeting different epitopes)

  • Alternatively, co-stain for FAM118B and known interacting partners such as coilin

  • FAM118B should colocalize with coilin in nuclear Cajal bodies

Implementing these validation controls ensures that experimental findings attributed to FAM118B are indeed specific and not the result of antibody cross-reactivity with unrelated proteins.

How can FAM118B be effectively studied in immunofluorescence microscopy experiments?

Immunofluorescence microscopy is a powerful technique for studying the subcellular localization of FAM118B, particularly its concentration in Cajal bodies. The following protocol has been optimized based on published research:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Both cancer cell lines and primary cells can be used, as FAM118B is expressed in a variety of cell types

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

  • For better visualization of FAM118B in Cajal bodies, extract cytosolic and nucleoplasmic proteins using Triton X-100 before fixation

  • For standard protocols, permeabilize fixed cells with 0.2% Triton X-100 in PBS for 5 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS for 1 hour at room temperature

  • Incubate with anti-FAM118B primary antibody (1:100-1:500 dilution) overnight at 4°C

  • Wash 3 times with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour at room temperature

  • For colocalization studies, co-stain with antibodies against Cajal body markers such as coilin (1:200) and SMN (1:200)

  • Wash 3 times with PBS, 5 minutes each

• Nuclear counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

  • Mount coverslips using anti-fade mounting medium

• Microscopy and analysis:

  • Visualize using a confocal microscope with appropriate laser lines

  • Focus on nuclear regions to identify Cajal bodies

  • In cells with intact Cajal bodies, FAM118B appears as concentrated foci that colocalize with coilin

  • Analyze the percentage of cells showing FAM118B localization in Cajal bodies

  • Quantify the number and size of FAM118B-positive foci per nucleus

• Advanced applications:

  • For studying the dynamics of FAM118B, consider using GFP-tagged FAM118B in live-cell imaging

  • To investigate FAM118B domain requirements, express truncation mutants and assess their localization patterns

  • For functional studies, combine immunofluorescence with RNA FISH to simultaneously visualize FAM118B and specific RNA targets

When interpreting immunofluorescence results, it's important to note that FAM118B localization is highly dependent on coilin. In coilin-depleted cells, FAM118B loses its concentrated localization in Cajal bodies and becomes diffusely distributed throughout the nucleoplasm .

What are common issues when detecting FAM118B and how can they be resolved?

Researchers may encounter several challenges when working with FAM118B antibodies. Here are common issues and their solutions:

• Weak or absent signal in Western blotting:

  • Increase antibody concentration (try 1:500 instead of 1:3000)

  • Extend primary antibody incubation time to overnight at 4°C

  • Increase protein loading to 50-75 μg per lane

  • Enhance signal using a more sensitive detection system (e.g., enhanced chemiluminescence)

  • Ensure sample preparation preserves FAM118B integrity by using fresh protease inhibitors

• Multiple bands or high background in Western blotting:

  • Increase blocking time or concentration (try 5% BSA instead of non-fat milk)

  • Reduce primary antibody concentration

  • Increase washing duration and number of washes

  • Use highly specific Prestige Antibodies® system (0.04-0.4 μg/mL)

  • Filter secondary antibody solution before use

• Inconsistent immunohistochemistry results:

  • Optimize antigen retrieval (compare citrate buffer pH 6.0 vs. TE buffer pH 9.0)

  • Ensure consistent fixation times across samples

  • Consider using automated staining platforms for better reproducibility

  • Titrate antibody concentration between 1:20-1:200 to determine optimal dilution

• Difficulty visualizing FAM118B in Cajal bodies:

  • Extract cytosolic and nucleoplasmic proteins using Triton X-100 before fixation to enhance visualization of FAM118B in Cajal bodies

  • Co-stain with coilin to identify Cajal bodies

  • Use confocal microscopy rather than widefield to better resolve nuclear structures

  • Ensure cells are in appropriate growth phase, as Cajal body formation can be cell cycle-dependent

• Cross-reactivity issues:

  • Validate antibody specificity using siRNA knockdown of FAM118B

  • Use affinity-purified antibodies specifically targeting FAM118B

  • Consider using antibodies raised against different epitopes of FAM118B to confirm findings

How can researchers optimize FAM118B knockdown experiments?

FAM118B knockdown experiments are valuable for studying its function, but require careful optimization:

• siRNA design and selection:

  • Target conserved regions of FAM118B mRNA

  • Design at least 3-4 different siRNA sequences

  • Avoid sequences with potential off-target effects

  • Published research has successfully reduced FAM118B protein levels to ~20% using specific siRNA sequences

• Transfection optimization:

  • Determine optimal cell density (typically 50-70% confluence at transfection)

  • Test different transfection reagents (lipofection vs. electroporation)

  • Optimize siRNA concentration (typically 20-100 nM)

  • Consider reverse transfection for hard-to-transfect cells

• Knockdown validation:

  • Assess FAM118B protein levels by Western blotting 48-72 hours post-transfection

  • Confirm reduction at mRNA level using qRT-PCR

  • Verify phenotypic effects by examining Cajal body morphology through immunofluorescence

• Controls:

  • Include non-targeting siRNA control

  • Use siRNA targeting a different gene (e.g., GAPDH) as a positive control

  • Include a rescue control by introducing siRNA-resistant FAM118B constructs

• Phenotypic analysis:

  • Examine multiple readouts: Cajal body morphology, SMN localization, splicing efficiency

  • For splicing assays, use both artificial splicing reporters and analyze endogenous gene splicing

  • Quantify the percentage of cells showing disrupted Cajal bodies (expect reduction from ~92% to ~2.5%)

  • Assess cell proliferation, as FAM118B depletion inhibits cell growth

• Rescue experiments:

  • Construct siRNA-resistant FAM118B by introducing silent mutations in the siRNA target region

  • Validate that expression levels match physiological FAM118B levels

  • Confirm rescue of phenotypes: Cajal body formation, splicing efficiency, cell proliferation

How does FAM118B research contribute to understanding nuclear organization and disease?

FAM118B research provides important insights into nuclear organization and potential disease mechanisms:

• Nuclear compartmentalization:

  • Studies of FAM118B enhance our understanding of how nuclear bodies like Cajal bodies form and function

  • FAM118B's role in maintaining Cajal body integrity highlights the importance of protein-protein interactions in organizing nuclear compartments

  • The finding that FAM118B depletion disrupts Cajal bodies suggests that proper nuclear organization depends on a network of interdependent factors

• RNA processing mechanisms:

  • FAM118B's involvement in pre-mRNA splicing reveals another layer of regulation in RNA processing

  • The connection between FAM118B, sDMA modification of SmD1, and snRNP assembly demonstrates how post-translational modifications can influence RNA metabolism

  • Quantitative analysis showing variable effects on different genes (10-63% reduction in spliced mRNA) suggests transcript-specific regulation

• Potential disease implications:

  • Given FAM118B's role in splicing, its dysfunction could contribute to diseases associated with splicing defects, such as certain neurodegenerative disorders

  • The inhibition of cell proliferation observed with FAM118B depletion suggests potential relevance to cancer biology

  • FAM118B's interaction with SMN protein, mutations in which cause spinal muscular atrophy, hints at possible connections to neuromuscular disorders

• Future research directions:

  • Investigate potential roles of FAM118B in additional RNA processing mechanisms beyond splicing

  • Explore tissue-specific functions of FAM118B, as it is expressed in various cell types

  • Examine whether FAM118B mutations or expression changes occur in human diseases

  • Develop more specific tools to modulate FAM118B function, such as small molecule inhibitors or enhancers

What emerging techniques could advance FAM118B research?

Several cutting-edge technologies could significantly enhance our understanding of FAM118B biology:

• CRISPR-Cas9 gene editing:

  • Generate FAM118B knockout cell lines for more complete functional studies

  • Create knock-in cell lines expressing tagged FAM118B at endogenous levels

  • Introduce specific mutations to study structure-function relationships

  • Develop CRISPR interference or activation systems for temporal control of FAM118B expression

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, PALM, SIM) to better visualize FAM118B within Cajal body structures

  • Live-cell imaging with fluorescently tagged FAM118B to study dynamics

  • Correlative light and electron microscopy to examine Cajal body ultrastructure in relation to FAM118B

  • Proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to FAM118B in living cells

• Proteomics approaches:

  • Comprehensive analysis of FAM118B interactome under various cellular conditions

  • Investigation of post-translational modifications on FAM118B itself

  • Quantitative proteomics to assess global changes in protein expression and modification following FAM118B manipulation

  • Targeted proteomics to monitor changes in symmetric dimethylarginine modification of specific proteins

• Transcriptome analysis:

  • RNA-seq to globally assess splicing changes upon FAM118B depletion or overexpression

  • Direct RNA sequencing to identify effects on RNA modifications

  • Single-cell RNA-seq to examine cell-to-cell variability in splicing efficiency

  • Spatial transcriptomics to investigate subcellular localization of affected transcripts

• Structural biology:

  • Cryo-EM or X-ray crystallography of FAM118B alone and in complex with interaction partners

  • NMR studies of the SIR2-like domain to understand its potential enzymatic function

  • Hydrogen-deuterium exchange mass spectrometry to map protein interaction surfaces

  • Molecular dynamics simulations to predict functional motions and binding interfaces

These emerging technologies could help resolve key questions about FAM118B function, such as the precise mechanism by which it regulates SmD1 symmetric dimethylarginine modification, its potential enzymatic activities, and its dynamic behavior within nuclear compartments.