FAM111B Antibody

What is FAM111B and what cellular functions does it serve in research contexts?

FAM111B is a 734 amino acid protein encoded by a gene located on human chromosome 11, which constitutes approximately 4% of human genomic DNA. FAM111B belongs to the FAM111 family and functions as a serine protease. This protein exists in multiple isoforms with molecular weights of approximately 135 kDa, 85 kDa, and 81 kDa .

FAM111B plays critical roles in:

• Cell cycle regulation, particularly during S-phase

• Cell proliferation control

• DNA replication processes

• Nuclear structure maintenance

Of particular interest to researchers, FAM111B has been identified as a direct target of p53, implicating it in cancer-related cellular pathways . Research has demonstrated its involvement in tendon contracture, myopathy, pulmonary fibrosis, and lung adenocarcinoma, making it an important target for studies across multiple disease contexts .

What cellular localization patterns does FAM111B typically display and how does this affect experimental design?

Immunofluorescence and fractionation studies have revealed that FAM111B displays a complex localization pattern that researchers must account for when designing experiments:

• FAM111B is present in both cytoplasm and nucleus, though the majority resides in the nuclear compartment (excluding nucleoli)

• Cell fractionation experiments show that FAM111B is largely soluble, with a fraction bound to insoluble components like chromatin or cellular membranes

• This binding to insoluble components increases when cells are blocked in early S-phase with hydroxyurea

• FAM111B interacts with components of nuclear pore complexes (NPCs), particularly nucleoporins SEC13 and NUP42, which contributes to its localization at the nuclear periphery

When designing experiments targeting FAM111B, researchers should consider these localization patterns, especially when interpreting subcellular fractionation data or immunofluorescence images. Different extraction protocols may be necessary to fully capture all FAM111B populations within the cell.

What are the recommended protocols for FAM111B immunohistochemistry in tissue samples?

For optimal FAM111B detection in tissue samples, researchers have successfully employed the following protocols:

Standard IHC Protocol:

• Deparaffinize tissue microarray sections

• Perform antigen retrieval using 1× citrate acid buffer

• Block endogenous peroxidase with 3% H₂O₂

• Apply primary anti-FAM111B antibody (1:100 dilution, such as Thermo PA5-28529) and incubate overnight at 4°C

• Rinse with 1× PBS

• Apply secondary antibody (e.g., goat anti-rabbit IgG H&L (HRP) at 1:400 dilution) and incubate overnight at 4°C

• Develop with diaminobenzidine (DAB) for 5 minutes

• Counterstain with hematoxylin for 10-15 seconds

Quantification Method:
When scoring FAM111B expression in tissue samples, a combined scoring system has been effectively used:

• Positive cell score: 0 (0%), 1 (1-24%), 2 (25-49%), 3 (50-74%), 4 (75-100%)

• Staining intensity score: 0 (no signal), 1 (light yellow), 2 (brownish yellow), 3 (dark brown)

• Calculate final IHC score by multiplying positive cell score by staining intensity score

This protocol has been validated in studies of bladder cancer tissues and provides reliable detection of FAM111B expression patterns.

How should researchers validate FAM111B antibody specificity to ensure reliable results?

Thorough validation of FAM111B antibodies is crucial for experimental success. Recommended validation approaches include:

• Positive and negative controls:

  • Use cell lines with known high FAM111B expression (e.g., T24 or EJ bladder cancer cell lines) as positive controls

  • Include non-malignant cell lines with lower expression (e.g., HCV-29 bladder epithelium) as comparative controls

  • Generate FAM111B-knockdown cell lines using validated shRNAs as negative controls

• Multiple detection methods:

  • Confirm antibody specificity across different applications (IHC, WB, IF)

  • Verify protein size on Western blots (expected bands at ~85 kDa and potentially at 135 kDa or 81 kDa for different isoforms)

• Antibody cross-validation:

  • Compare staining patterns using antibodies from different sources or recognizing different epitopes

  • For example, compare results between antibodies targeting different regions of FAM111B (such as Thermo PA5-28529 versus Proteintech 20552-1-AP)

• Genetic approaches:

  • Use CRISPR/Cas9 knockout cells as definitive negative controls

  • Complement with FAM111B overexpression models to confirm signal specificity

Implementing these validation steps significantly increases confidence in experimental findings involving FAM111B antibodies.

How can FAM111B antibodies be effectively used to study cancer progression mechanisms?

FAM111B antibodies have proven valuable in investigating cancer progression mechanisms through several methodological approaches:

Tumor Tissue Analysis:

Functional Studies in Cancer Models:
When studying FAM111B's role in cancer, researchers have successfully:

• Created FAM111B-knockdown cancer cell models using lentiviral shRNA delivery

• Assessed the effects on:

  • Cell proliferation (using cell count assays)

  • Migration (using wound-healing and transwell assays)

  • Apoptosis (using Annexin V-APC staining and flow cytometry)

• Confirmed in vivo relevance through xenograft tumor models

Mechanistic Investigations:

• Human apoptosis antibody arrays have been used to detect the expression levels of 43 human apoptosis markers following FAM111B knockdown

• This approach has revealed downstream pathways affected by FAM111B expression

For researchers interested in cancer biology, these methodologies provide a framework for investigating FAM111B's role in specific cancer types and contexts.

What techniques can be used to investigate FAM111B's interactions with nuclear pore complexes?

Recent research has revealed that FAM111B interacts with nuclear pore complexes (NPCs), suggesting important functions at the nuclear periphery. To study these interactions, researchers have employed the following techniques:

Mass Spectrometry-Based Interactome Analysis:

• Immunoprecipitation of FAM111B followed by mass spectrometry has identified interactions with NPC components, particularly nucleoporins SEC13 and NUP42

• This approach provides an unbiased profile of protein interactions

Co-Immunoprecipitation Confirmation:

• Following mass spectrometry identification, co-IP experiments with FLAG-FAM111B have confirmed specific interactions with nucleoporins

• This technique allows validation of individual protein-protein interactions

Localization Studies:

• Detailed immunofluorescence microscopy after extracting soluble proteins with detergent reveals FAM111B localization patterns at the nuclear periphery

• Three distinct patterns have been observed and quantified: pan nuclear, peripheral enriched, and exclusively peripheral

Mutant Analysis:

• Expression of FAM111B variants (wild-type, protease-dead, or disease-associated mutants like Q430P) as FLAG-tagged proteins allows comparison of their localization patterns

• This approach helps determine which domains are essential for NPC interaction

These methodologies provide a comprehensive toolkit for researchers investigating FAM111B's role at the nuclear envelope and its functional interactions with nuclear pore complexes.

What factors affect FAM111B detection in experimental systems and how can they be controlled?

Several factors can significantly impact FAM111B detection in research applications:

Cell Cycle Dependence:

• FAM111B protein levels are strongly modulated by cell cycle position, with enrichment in S-phase cells showing characteristic PCNA replication foci

• Researchers should consider cell synchronization or cell cycle analysis when comparing FAM111B levels between samples

• Flow cytometry with BrdU staining can help correlate FAM111B expression with specific cell cycle phases

Extraction Conditions:

• Different fractionation protocols may yield varying results due to FAM111B's distribution between soluble and insoluble cellular compartments

• More stringent extraction conditions may be needed to fully solubilize FAM111B associated with chromatin or membranes

• Hydroxyurea treatment affects FAM111B's association with insoluble components, potentially affecting extraction efficiency

Antibody Selection:

• Different commercial antibodies target distinct epitopes within FAM111B

• Some antibodies may preferentially detect specific isoforms or post-translationally modified forms

• Cross-validation with multiple antibodies is recommended for critical experiments

Fixation Methods:

• For immunofluorescence or IHC, fixation protocols can influence antibody accessibility to FAM111B epitopes

• Optimization of fixation conditions (formaldehyde concentration, duration, temperature) may be necessary for specific applications

Controlling these variables through careful experimental design and appropriate controls enables more reliable and reproducible FAM111B detection across different experimental systems.

How should researchers interpret conflicting data regarding FAM111B's role in different disease contexts?

When encountering conflicting data regarding FAM111B's role in different diseases, researchers should consider several methodological approaches:

Context-Specific Functions:

Disease-Specific Association Analysis:

• While FAM111B mutations cause hereditary fibrosing poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis (POIKTMP), they are not associated with systemic sclerosis despite clinical similarities

• This highlights the importance of direct genetic analysis rather than relying on phenotypic similarities alone

Reconciliation Strategies:

• Molecular mechanism analysis: Determine if FAM111B's protease activity has different substrates in different tissues

• Pathway analysis: Investigate if FAM111B interfaces with different signaling pathways depending on the cellular context

• Mutation-specific effects: Distinguish between loss-of-function, gain-of-function, or neomorphic effects of different FAM111B mutations

• Expression level considerations: Assess whether FAM111B's effects are dose-dependent, with different outcomes at low versus high expression levels

When interpreting contradictory findings, researchers should also consider technical differences between studies, including antibody specificity, detection methods, and experimental systems used.

What emerging methodologies could advance our understanding of FAM111B's biological functions?

Several cutting-edge methodologies hold promise for deepening our understanding of FAM111B biology:

Protease Substrate Identification:

• Since FAM111B functions as a serine protease, identifying its substrates is crucial

• Quantitative proteomics comparing wild-type and protease-dead mutants could reveal FAM111B-dependent proteolytic events

• TAILS (Terminal Amine Isotopic Labeling of Substrates) methodology could specifically identify FAM111B cleavage sites in target proteins

High-Resolution Imaging:

• Super-resolution microscopy techniques could better define FAM111B's dynamic localization during cell cycle progression

• Live-cell imaging with fluorescently-tagged FAM111B would reveal its real-time behavior during DNA replication and mitosis

Single-Cell Analysis:

• Single-cell RNA-seq combined with protein analysis could reveal cell-to-cell variability in FAM111B expression and function

• This would be particularly valuable in heterogeneous tumor samples

Structural Biology:

• Determining the crystal structure of FAM111B's protease domain would facilitate understanding of how disease-associated mutations affect its activity

• Structure-guided drug design could eventually target FAM111B in cancer contexts

Genome-Wide CRISPR Screens:

• Synthetic lethality screens in FAM111B-dependent cancer cell lines could identify potential therapeutic vulnerabilities

• Genetic interaction mapping could place FAM111B within broader cellular pathways

These advanced methodologies represent promising directions for researchers seeking to elucidate FAM111B's complex biological functions and disease associations.

How might FAM111B antibodies be utilized in translational research applications?

FAM111B antibodies have significant potential for translational research applications:

Prognostic Biomarker Development:

• Given the correlation between FAM111B expression and clinical outcomes in bladder cancer and lung adenocarcinoma, standardized IHC protocols could be developed for prognostic assessment

• A quantitative IHC scoring system has already shown promise in correlating FAM111B levels with survival outcomes

Therapeutic Response Prediction:

• FAM111B expression patterns might predict response to specific cancer therapies

• Antibody-based tissue analysis before and after treatment could help identify responder versus non-responder molecular signatures

Companion Diagnostics:

• If FAM111B-targeting therapeutics are developed, antibody-based assays could identify patients most likely to benefit

• Different antibodies recognizing specific FAM111B conformations or mutations might be valuable for patient stratification

Liquid Biopsy Applications:

• Detection of FAM111B protein in circulating tumor cells or extracellular vesicles might serve as a minimally invasive biomarker

• This would require highly sensitive and specific antibodies optimized for such applications

Monitoring Treatment Effects:

• In diseases associated with FAM111B mutations, antibodies distinguishing between wild-type and mutant forms could help monitor disease progression or treatment efficacy

These translational applications represent promising avenues for moving FAM111B research from basic science into clinical utility.