FAM135B Antibody

What is FAM135B and why is it significant for research?

FAM135B (Family with sequence similarity 135 member B) is a novel DNA damage response (DDR) regulator that has garnered significant attention in cancer research. It functions by physically interacting with TIP60 (Tat-interactive protein 60-kDa) and enhancing its histone acetyltransferase (HAT) activity. This interaction plays a crucial role in maintaining genomic integrity through the TIP60/ATM (ataxia-telangiectasia-mutated) axis-mediated DNA damage response . FAM135B has been identified as a potential driver gene in oesophageal squamous cell carcinoma (ESCC) and is frequently mutated and amplified in ESCC patients . Its ability to protect cancer cells from genotoxic stress in both in vitro and in vivo models makes it a compelling target for understanding chemotherapy and radiotherapy resistance mechanisms .

What are the validated applications for FAM135B antibody?

FAM135B antibody has been validated for multiple research applications based on the testing data. The primary applications include:

For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, with an alternative option of citrate buffer pH 6.0 . These validated applications provide researchers with reliable methods for detecting and studying FAM135B expression across various experimental models.

How does FAM135B antibody perform in different cellular compartments?

FAM135B has been localized in both nuclear and cytoplasmic compartments, with its interaction with TIP60 primarily occurring in the nucleus . When performing immunofluorescence studies, researchers should expect to detect FAM135B signals in both compartments, with particularly strong co-localization with TIP60 in the nuclear region. This dual localization is consistent with FAM135B's roles in both DNA damage response (nuclear) and potential cytoplasmic signaling functions through the GRN/AKT/mTOR pathway as previously reported . When designing experiments to detect FAM135B, researchers should consider cell permeabilization protocols that allow antibody access to both compartments, particularly when studying its role in DNA damage response mechanisms.

How does FAM135B expression change during DNA damage and repair processes?

FAM135B exhibits a distinctive expression pattern during DNA damage and repair. Upon exposure to genotoxic agents such as cisplatin (CDDP) and bleomycin (BLM), FAM135B protein levels significantly decrease, though not in a strictly dose- or time-dependent manner . This reduction occurs at the protein level rather than the transcriptional level, as mRNA levels remain relatively unchanged during treatment .

During the DNA repair period (after removal of the genotoxic stress), FAM135B levels gradually increase again . This dynamic regulation appears to be controlled through proteasomal degradation rather than transcriptional regulation. The pattern suggests FAM135B plays different roles before damage (maintaining TIP60-ATM reservoir), during damage (released from TIP60 to allow the complex to function), and during repair (gradually returning to maintain the pre-assembly reservoir) .

This expression pattern is critical for researchers to consider when designing time-course experiments examining FAM135B's role in DNA damage response, as sampling at different timepoints will yield different expression profiles.

What is the molecular mechanism of FAM135B interaction with TIP60?

FAM135B physically interacts with TIP60 through a specific domain interaction that has been carefully characterized. Co-immunoprecipitation assays have confirmed that endogenous FAM135B and TIP60 can immunoprecipitate each other . This interaction has been further validated using exogenous expression systems with tagged proteins (FAM135B-FLAG) and through GST pull-down assays with purified recombinant proteins .

Domain mapping experiments have revealed that FAM135B specifically binds to the chromodomain (ChD) of TIP60 . This was determined using various TIP60 fragments in pull-down assays:

• Full-length TIP60 (1-513 aa): Interaction detected

• Δ1 fragment (1-258 aa, containing chromodomain): Interaction detected

• Δ2 fragment (69-290 aa, Zinc finger domain): No interaction

• Δ3 fragment (158-395 aa, Zinc finger and acetyl-CoA-binding domains): No interaction

• Δ4 fragment (285-513 aa, acetyl-CoA-binding and NR domains): No interaction

This specific interaction with the chromodomain is particularly significant as it suggests a regulatory mechanism whereby FAM135B may influence TIP60's ability to recognize and bind to modified histones during DNA damage response.

How can researchers detect changes in FAM135B-TIP60-ATM complex formation?

To effectively study the dynamic formation and regulation of the FAM135B-TIP60-ATM complex, researchers should employ a multi-method approach:

• Co-immunoprecipitation (Co-IP): The primary method validated for detecting these interactions. Researchers can immunoprecipitate with antibodies against any of the three proteins (FAM135B, TIP60, or ATM) and then blot for the other components .

• Proximity Ligation Assay (PLA): This technique has been successfully used to detect protein-protein interactions in the study and provides spatial information about where in the cell these interactions occur .

• GST Pull-down Assays: For confirming direct physical interactions between purified components, particularly useful for domain mapping studies .

• Immunofluorescence Co-localization: While less quantitative than the above methods, IF can provide visual confirmation of the spatial overlap between FAM135B and TIP60, primarily in the nucleus .

When designing experiments to monitor changes in complex formation under different conditions (normal vs. DNA damage), researchers should consider time-course experiments, as the dynamic association and dissociation of FAM135B from TIP60 occurs over time following DNA damage induction.

What are the optimal conditions for FAM135B antibody use in detecting DNA damage response?

When designing experiments to study FAM135B in DNA damage response contexts, researchers should consider several critical parameters:

• Timing of sample collection: FAM135B protein levels significantly decrease following DNA damage but gradually increase during repair. For comprehensive analysis, collect samples at multiple timepoints: before damage (baseline), immediately after damage (early response), and during repair phase (6h, 12h, 24h post-damage) .

• DNA damage induction protocols: Validated agents include bleomycin (BLM), cisplatin (CDDP), etoposide (ETO), and γ-irradiation (IR) . The optimal concentrations reported were 10-μM BLM for 12 hours to induce DNA damage, followed by media replacement to allow repair .

• Controls: Include both negative controls (untreated cells) and positive controls (cells with known DNA damage markers such as γH2AX) .

• Complementary markers: Always include established DNA damage markers like γH2AX and 53BP1 in parallel experiments to correlate FAM135B changes with canonical DNA damage responses .

• Antibody validation: Confirm antibody specificity using FAM135B overexpression or knockdown controls to ensure signals detected are specific to FAM135B .

These optimized conditions will help ensure reliable detection of FAM135B's dynamic changes during DNA damage response and repair processes.

How should researchers interpret contradictory FAM135B antibody results in different cancer models?

When facing contradictory FAM135B antibody results across different cancer models, researchers should consider several factors that might explain these discrepancies:

• Tissue-specific expression patterns: FAM135B expression and function may vary significantly across tissue types. The antibody has been validated in neural cells (C6, Neuro-2a), kidney tissue, and brain tissue , but may show different patterns in other tissues.

• Cancer-specific alterations: FAM135B is frequently mutated and amplified in ESCC , but its status in other cancers may differ. Mutations might affect antibody epitope recognition.

• DDR pathway status: The functionality of the DDR pathway varies across cancer types. Since FAM135B interacts with the TIP60-ATM axis, cancers with alterations in these pathways may show different FAM135B behavior.

• Technical considerations:

  • Antibody concentration: Different optimal dilutions may be needed for different tissues (1:500-1:2000 for WB, 1:50-1:100 for IHC)

  • Antigen retrieval method: TE buffer pH 9.0 is recommended, but citrate buffer pH 6.0 is an alternative

  • Fixation methods can significantly impact epitope accessibility

To resolve contradictions, researchers should:

• Validate findings using multiple antibodies or detection methods

• Combine protein detection with mRNA analysis

• Use genetic modulation (overexpression/knockdown) to confirm specificity

• Consider the temporal dynamics of FAM135B during DNA damage and repair processes

What controls should be included when studying FAM135B's role in DNA repair pathways?

To ensure robust and reproducible findings when studying FAM135B's role in DNA repair pathways, researchers should include these essential controls:

• Expression validation controls:

  • FAM135B overexpression system (verified by Western blot)

  • FAM135B knockdown (siRNA or shRNA, verified by Western blot)

  • These manipulations provide clear reference points for antibody specificity and function

• DNA damage induction controls:

  • Positive controls: Cells treated with established DNA damaging agents (BLM, CDDP, ETO, IR)

  • Time-course controls: Samples collected at multiple timepoints to capture the dynamic nature of FAM135B response

  • γH2AX and 53BP1 staining as established DNA damage markers to confirm damage induction

• Repair pathway-specific controls:

  • For homologous recombination (HR) studies: Include RAD51 foci assessment

  • For non-homologous end-joining (NHEJ) studies: Include DNA-PKcs or Ku70/80 assessment

  • FAM135B has been shown to promote both HR and NHEJ repair pathways

• Functional assay controls:

  • Comet assay controls: Include both positive (damaged) and negative (undamaged) reference samples

  • Colony formation assay: Include standard curves with known cell numbers

  • Flow cytometry: Include single-color controls for compensation

• Interaction controls for FAM135B-TIP60-ATM studies:

  • IgG control for immunoprecipitation experiments

  • GST-only control for pull-down assays

  • Peptide competition controls for antibody specificity

By incorporating these comprehensive controls, researchers can establish the specificity and reliability of their findings regarding FAM135B's role in DNA damage repair pathways.

What are the recommended protocols for detecting FAM135B in tissue samples versus cell cultures?

Different sample types require optimized protocols for effective FAM135B detection:

For Tissue Samples (IHC/IF):

• Fixation: 10% neutral buffered formalin is recommended for preserving FAM135B without compromising epitope integrity.

• Antigen Retrieval: The validated method is heat-mediated antigen retrieval with TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 can be used .

• Blocking: 5-10% normal serum in PBS for 1 hour at room temperature to reduce non-specific binding.

• Primary Antibody: Use at 1:50-1:100 dilution for IHC applications .

• Detection: DAB chromogen system for IHC or fluorophore-conjugated secondary antibodies for IF.

• Counterstaining: Hematoxylin for IHC or DAPI for nuclear counterstaining in IF.

• Positive Control Tissues: Mouse brain tissue and human kidney tissue have been validated for positive staining .

For Cell Cultures (WB/IF):

• Cell Lysis: Standard RIPA buffer supplemented with protease inhibitors is effective for extracting total FAM135B.

• Protein Quantification: Bradford or BCA assay to ensure equal loading.

• SDS-PAGE: 8-10% gel recommended due to FAM135B's molecular weight.

• Transfer: Standard wet transfer protocols are suitable.

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody: Use at 1:500-1:2000 dilution for WB applications .

• Validated Cell Lines: C6 cells and Neuro-2a cells have demonstrated reliable detection .

Key Differences to Consider:

• Tissue samples require more extensive antigen retrieval compared to cell cultures

• Antibody concentration is typically higher for tissue applications (1:50-1:100) than for cell culture applications (1:500-1:2000)

• Nuclear localization of FAM135B may require permeabilization optimization in IF protocols

These optimized protocols ensure reliable detection of FAM135B across different experimental systems while minimizing background and non-specific signals.

How can researchers effectively monitor FAM135B dynamics during DNA damage and repair?

To effectively monitor FAM135B dynamics during DNA damage and repair processes, researchers should implement a comprehensive experimental approach that captures the protein's behavior throughout the response:

• Time-course experimental design:

  • Baseline (untreated): Establish normal FAM135B levels

  • Acute damage phase: Sample immediately after damage induction

  • Early repair phase: 2-6 hours post-damage

  • Mid repair phase: 12 hours post-damage

  • Late repair phase: 24 hours post-damage

• Protein level monitoring techniques:

  • Western blot: Quantify total FAM135B protein level changes over time

  • Immunofluorescence: Track subcellular localization changes

  • Proteasome inhibitor experiments: Add MG132 to determine if protein degradation mechanisms are involved in FAM135B reduction after DNA damage

• Interaction dynamics assessment:

  • Co-immunoprecipitation at different timepoints to track FAM135B-TIP60 interaction changes

  • Proximity ligation assay (PLA) to visualize intact complexes within cells

  • ChIP assays to monitor chromatin association of TIP60-ATM after FAM135B release

• Damage-repair correlation markers:

  • γH2AX foci: Track in parallel with FAM135B to correlate with damage resolution

  • 53BP1 foci: Monitor recruitment and resolution kinetics

  • Comet assay: Quantify DNA damage at matching timepoints

• Experimental controls:

  • FAM135B overexpression and knockdown conditions to establish causality

  • Multiple DNA damaging agents (BLM, CDDP, ETO, IR) to ensure phenomenon is not agent-specific

These methodological approaches will provide a comprehensive profile of FAM135B dynamics throughout the DNA damage response and repair processes, allowing researchers to accurately characterize its role in these critical cellular functions.

What are the challenges in using FAM135B antibody for in vivo experiments?

Researchers using FAM135B antibody for in vivo experiments face several technical and biological challenges that require careful consideration:

• Tissue-specific expression variability:

  • FAM135B expression levels vary significantly across tissues, requiring optimization of antibody concentration for each tissue type

  • Validated positivity has been demonstrated in mouse brain tissue and human kidney tissue, but other tissues may require additional validation

  • The FAM135B transgenic mouse model shows tissue-specific effects that may complicate interpretation

• Fixation and processing challenges:

  • Overfixation can mask the FAM135B epitope, requiring careful titration of fixation times

  • Different fixatives (paraformaldehyde vs. formalin) may yield different results

  • Antigen retrieval with TE buffer pH 9.0 is critical; citrate buffer pH 6.0 is an alternative but may yield different staining intensity

• Background and specificity concerns:

  • Endogenous mouse immunoglobulins can interact with detection antibodies, producing false positives

  • Autofluorescence, particularly in tissues like liver, can complicate IF analysis

  • Mouse-on-mouse detection issues when using mouse monoclonal antibodies on mouse tissues

• Dynamic regulation during experimental interventions:

  • FAM135B levels fluctuate significantly following DNA damage, making timing of tissue collection critical

  • Radiation treatment (as used in the FAM135Btg mouse model) affects FAM135B expression patterns, requiring careful experimental design with appropriate controls

• Quantification challenges:

  • IHC scoring systems must be standardized across samples

  • The dual nuclear/cytoplasmic localization complicates automated image analysis approaches

  • Correlation with functional outcomes requires parallel γH2AX staining

To address these challenges, researchers should:

• Include wild-type controls alongside FAM135Btg mice

• Implement rigorous tissue processing protocols with standardized fixation times

• Use tyramide signal amplification for enhanced sensitivity in tissues with low expression

• Consider phospho-specific antibodies to track activation state rather than just total protein

• Validate findings using multiple detection methods (IHC, IF, WB from tissue lysates)

These approaches will help overcome the inherent challenges in studying FAM135B in vivo while generating reliable and reproducible results.

How might FAM135B antibodies be utilized in studying chemotherapy and radiation resistance?

FAM135B's established role in protecting cancer cells from genotoxic stress positions it as a promising biomarker and potential therapeutic target for addressing therapy resistance. Researchers can utilize FAM135B antibodies in several innovative approaches:

• Predictive biomarker development:

  • Tissue microarray studies correlating FAM135B expression with treatment outcomes

  • IHC analysis of FAM135B in pre- and post-treatment biopsies to evaluate adaptive responses

  • A preliminary study has already shown that FAM135B expression is significantly higher in platinum-resistant patients compared to sensitive responders

• Resistance mechanism characterization:

  • Monitor FAM135B-TIP60-ATM complex dynamics before and after treatment

  • Track FAM135B's subcellular redistribution during acquired resistance development

  • Combine with γH2AX staining to correlate FAM135B levels with DNA damage resolution efficiency

• Combination therapy development:

  • Screen for compounds that modulate FAM135B-TIP60 interaction

  • Test synthetic lethality approaches combining FAM135B inhibition with other DDR pathway inhibitors

  • FAM135B has been proposed as a potential target for synthetic lethality approaches

• Radiation sensitivity studies:

  • Use the validated FAM135Btg mouse model to further characterize radiation response

  • Measure γH2AX clearance kinetics in FAM135B-high versus FAM135B-low tumors

  • The oesophageal model from FAM135Btg mice shows differential radiation sensitivity that could be further explored

• Patient stratification approaches:

  • Develop standardized IHC scoring systems for FAM135B to guide treatment selection

  • Correlate FAM135B levels with specific mutation profiles to identify vulnerable patient subgroups

  • Integrate FAM135B status with other DDR biomarkers for comprehensive resistance profiling

These research directions leverage the specificity of FAM135B antibodies to address the significant clinical challenge of therapy resistance, potentially leading to more effective treatment strategies for cancer patients.

What are the considerations for developing phospho-specific FAM135B antibodies?

While current research focuses on total FAM135B levels, developing phospho-specific FAM135B antibodies would provide deeper insights into its regulation and function. Researchers should consider these key aspects:

• Potential phosphorylation sites identification:

  • In silico prediction suggests multiple potential phosphorylation sites in FAM135B

  • Proteomic analysis of FAM135B under normal and DNA damage conditions would identify which sites undergo dynamic modification

  • Key candidates would include sites regulated by DNA damage-responsive kinases (ATM, ATR, DNA-PK)

  • Priority should be given to sites that affect protein stability, as FAM135B levels decrease following DNA damage

• Technical considerations for antibody development:

  • Synthesize phosphopeptides corresponding to predicted sites

  • Ensure peptide design accounts for surrounding amino acids that affect epitope recognition

  • Use carrier proteins to improve immunogenicity of small phosphopeptides

  • Implement extensive validation with phosphatase treatment controls

• Validation experiments necessary for phospho-antibodies:

  • Phosphomimetic mutants (S/T to D/E) and phospho-dead mutants (S/T to A)

  • Lambda phosphatase treatment controls

  • Kinase inhibitor treatments to confirm pathway specificity

  • Parallel detection with pan-FAM135B antibodies to calculate phosphorylation stoichiometry

• Functional significance assessment:

  • Monitor phosphorylation status during DNA damage and repair kinetics

  • Correlate phosphorylation with FAM135B-TIP60 interaction changes

  • Examine if phosphorylation precedes FAM135B degradation after DNA damage

  • Test if phosphorylation affects FAM135B's ability to enhance TIP60's HAT activity

• Potential applications of phospho-FAM135B antibodies:

  • More precise biomarkers for activation state rather than just expression

  • Better understanding of upstream regulatory pathways

  • Identification of druggable kinase targets that indirectly affect FAM135B function

  • Correlation of FAM135B phosphorylation status with therapy resistance profiles

These considerations provide a roadmap for developing and validating phospho-specific FAM135B antibodies that would significantly advance our understanding of this protein's regulatory mechanisms in DNA damage response.