FTSJ1 Antibody

What is FTSJ1 and what cellular functions does it perform?

FTSJ1 is a tRNA-specific 2′-O-methyltransferase enzyme primarily responsible for methylation at positions 32 and 34 of specific tRNAs. It plays a critical role in tRNA modification, which is essential for accurate decoding during protein translation . FTSJ1 catalyzes the formation of 2′-O-methylation (Nm) on different tRNA substrates, with m1G37 serving as a prerequisite for Nm34 formation .

The enzyme is predominantly localized in the cytoplasm with smaller amounts detected in the nucleus, as confirmed by both immunolabeling and nuclear/cytosol fractionation assays . FTSJ1 functions in complex with WDR6, which serves as the tRNA-binding component while FTSJ1 binds S-adenosyl-L-methionine (SAM) as the methyl donor to support catalytic activity .

Recent studies have revealed that FTSJ1 is involved in both neurological development and cancer progression, making it an important target for research in multiple fields .

What are the recommended protocols for using FTSJ1 antibodies in Western blotting?

When performing Western blot analysis with FTSJ1 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Extract total protein from your cell lines or tissues using standard lysis buffers. Research indicates successful use of FTSJ1 antibodies with HCC1937, MDA-MB-231, and 4T1 cell lines .

• Antibody selection: For optimal results, validated antibodies such as Abcam's ab227259 have been successfully employed in published research .

• Quantification approach: For accurate quantitative analysis, employ software such as Image Lab 6.0.1 (Bio-Rad) to measure and analyze blots' grayscale values .

• Controls: Always include appropriate positive and negative controls. For knockdown experiments, verify FTSJ1 expression reduction using Western blot before proceeding with functional assays .

• Validation: When assessing knockdown efficiency, compare the protein expression levels between control and knockdown samples by normalizing to appropriate housekeeping proteins .

How can FTSJ1 antibodies be used in immunohistochemistry and immunofluorescence applications?

For immunohistochemical (IHC) and immunofluorescence (IF) staining using FTSJ1 antibodies, the following protocol has been validated in clinical specimens:

• Sample preparation: Fix tissues in formalin and embed in paraffin. Cut sections at 4-5 μm thickness and mount on slides.

• Antigen retrieval: After deparaffinization and rehydration, perform antigen retrieval, then block endogenous peroxidase activity with 3% H₂O₂ solution for 10 minutes .

• Blocking and primary antibody: Block with 5% goat serum for 1 hour, then apply primary FTSJ1 antibodies (e.g., Abcam, ab227259) and incubate overnight at 4°C .

• Detection system: For IHC, use Two-Step IHC reagents and 3,3-diaminobenzidine (DAB) solution according to manufacturer's instructions. Counterstain with Harris modified hematoxylin .

• Scoring system: Assess staining intensity using a four-category classification: negative (0), weak (1), moderate (2), and strong (3). Evaluate percentage of positive cells using a scale of 0 (0–5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%) .

For multi-staining immunofluorescence applications, FTSJ1 antibodies can be combined with other markers such as CD8 and perforin to study correlations between FTSJ1 expression and immune cell infiltration in tumor tissues .

How can researchers effectively study FTSJ1-WDR6 interactions using antibodies?

The interaction between FTSJ1 and WDR6 is critical for the methyltransferase activity of FTSJ1. To study this interaction:

• Co-immunoprecipitation approach: Express FTSJ1 with a C-terminal Flag tag (FTSJ1-Flag) and WDR6 with an HA tag (WDR6-HA) in an appropriate cell line such as HEK293T. Perform reciprocal co-immunoprecipitation by pulling down with anti-Flag antibody and detecting WDR6-HA, and vice versa .

• Domain mapping: The methyltransferase (MTase) catalytic domain of FTSJ1 (approximately residues 19-220) is important for interaction studies. Design truncation mutants to identify specific interacting regions .

• Functional validation: After identifying interactions, validate the functional significance by reconstituting the 2′-O-methylation activity of the FTSJ1-WDR6 complex in vitro. This requires purified components and appropriate tRNA substrates with pre-existing modifications .

• Controls and specificity: Include negative controls such as unrelated proteins tagged with the same epitopes to confirm specificity of the interaction. In published research, THADA was not detected as an interacting partner, providing a potential negative control .

What methodological approaches should be used to study FTSJ1's role in cancer progression?

To investigate FTSJ1's role in cancer progression, particularly in triple-negative breast cancer (TNBC), consider these methodological approaches:

• Expression analysis in clinical samples: Use FTSJ1 antibodies for IHC staining of TNBC tissue microarrays to correlate expression levels with clinical outcomes. High FTSJ1 expression has been associated with poor prognosis in TNBC patients .

• Functional studies using knockdown models:

  • Generate stable FTSJ1 knockdown cell lines using appropriate TNBC cell models (HCC1937, MDA-MB-231)

  • Validate knockdown efficiency by Western blot

  • Assess effects on proliferation, migration, and apoptosis using standard assays

  • Perform in vivo studies using xenograft tumor mouse models

• Immune infiltration analysis:

  • Analyze correlation between FTSJ1 expression and tumor-infiltrating immune cells using ssGSEA algorithm in public datasets like TCGA-BRCA

  • Perform flow cytometry analysis of CD8+ and CD4+ T cells in tumor infiltration in FTSJ1 knockdown versus control tumors

  • Use multi-staining immunofluorescence to examine relationships between FTSJ1 expression and CD8+ T cell infiltration in tumor tissues

• T cell killing assays:

  • Establish co-culture systems using fluorescent-labeled tumor cells and activated T cells

  • Assess how FTSJ1 knockdown affects tumor cell sensitivity to T cell-mediated cytotoxicity

  • Compare results between knockdown, overexpression, and control conditions

What controls and validation methods are essential when studying FTSJ1 knockdown effects?

When studying FTSJ1 knockdown effects, implement these critical controls and validation methods:

• Knockdown validation at protein level: Confirm reduction of FTSJ1 protein expression by Western blot analysis. Quantify using band intensity analysis software to determine knockdown efficiency .

• Validation at RNA level: For mutations affecting mRNA stability or when studying nonsense-mediated decay (NMD), treat cells with cycloheximide to inhibit NMD before RNA extraction. Use RT-PCR with specific primers to amplify FTSJ1 cDNAs (e.g., Forward: 5'-GGCAGTTGACCTGTGTGCAGC-3'; Reverse: 5'-CCCTCTAGGTCCAGTGGGTAAC-3') .

• Functional validation: Confirm that knockdown affects the expected methyltransferase activity by analyzing tRNA modification status using techniques like RiboMethSeq .

• Phenotypic rescue experiments: Restore FTSJ1 expression in knockdown cells to demonstrate that observed phenotypes are specifically due to FTSJ1 depletion rather than off-target effects .

• Multiple knockdown methods: Use different approaches (siRNA, shRNA, CRISPR-Cas9) to validate that phenotypes are consistent regardless of the knockdown method used .

How can researchers effectively investigate FTSJ1's role in tRNA modification using antibodies?

To study FTSJ1's role in tRNA modification using antibodies:

• Immunoprecipitation of FTSJ1-tRNA complexes:

  • Use validated FTSJ1 antibodies to immunoprecipitate the protein along with bound tRNAs

  • Extract and sequence associated RNAs to identify specific tRNA substrates

  • Confirm the presence of known targets like tRNAPhe(GAA)

• In vitro reconstitution assays:

  • Express and purify FTSJ1 along with its cofactor WDR6

  • Perform in vitro methylation assays using synthesized or purified tRNAs as substrates

  • Verify that m1G37 is present in substrates as it's a prerequisite for Nm34 formation by FTSJ1-WDR6

• Analysis of modification interdependence:

  • Study the hierarchy of modifications at positions 32, 34, and 37 of specific tRNAs

  • Use antibodies to pull down FTSJ1 from cells with various tRNA modification enzyme knockdowns to understand sequential modification patterns

• Translation efficiency studies:

  • After confirming FTSJ1 knockdown with antibodies, assess codon-specific translation efficiency

  • Focus on UUU codons which are specifically affected by loss of FTSJ1-mediated tRNAPhe(GAA) modification

What methodological considerations apply when using FTSJ1 antibodies in neuronal research?

When using FTSJ1 antibodies in neuronal research, consider these methodological approaches:

• Neuronal differentiation studies:

  • Generate FTSJ1-depleted human neuronal progenitor cells (NPCs)

  • Monitor differentiation into neurons using appropriate markers

  • Analyze neurite morphology, focusing on spine length and thickness

  • Compare results with animal models like Drosophila to confirm conservation of phenotypes

• Transcriptome analysis:

  • After confirming FTSJ1 depletion with antibodies, perform RNA-seq analysis

  • Focus on genes previously associated with intellectual disability that are deregulated in FTSJ1-depleted cells

  • Analyze changes in miRNA populations that might cross-regulate with key mRNA targets

• Patient-derived cell studies:

  • Obtain cells from intellectual disability patients with FTSJ1 mutations

  • Perform comprehensive RiboMethSeq analysis to map ribose methylation on all tRNAs

  • Compare with normal controls to identify differential tRNA modification patterns

• Functional assays:

  • Use model organisms like Drosophila to study long-term memory deficits associated with FTSJ1 depletion

  • Correlate these findings with molecular and morphological changes observed in human neuronal models

What are the most common issues when using FTSJ1 antibodies and how can they be addressed?

When working with FTSJ1 antibodies, researchers may encounter several challenges:

• Non-specific binding: Ensure proper blocking (5% goat serum has been validated) and antibody dilution. Implement stringent washing steps between incubations .

• Inconsistent staining intensity: For IHC applications, optimize antigen retrieval methods and standardize DAB development time. Use a consistent scoring system to evaluate staining intensity and percentage of positive cells .

• Low signal in Western blots: Optimize protein extraction protocols to ensure FTSJ1 is adequately solubilized. Since FTSJ1 is predominantly cytoplasmic with some nuclear localization, ensure your extraction method efficiently captures both fractions .

• Variability in co-immunoprecipitation: When studying FTSJ1-WDR6 interactions, optimize buffer conditions and ensure appropriate epitope tag placement to avoid interfering with protein-protein interactions .

• Batch-to-batch variability: Validate each new antibody lot against known positive controls. Consider creating standard reference samples that can be used across experiments to normalize for antibody performance variations.

How can researchers validate that their FTSJ1 antibody is detecting the correct target?

To confirm FTSJ1 antibody specificity:

• Knockdown/knockout controls: Generate FTSJ1 knockdown or knockout cell lines and confirm reduced or absent signal in Western blot, IHC, or immunofluorescence applications .

• Overexpression validation: Express tagged FTSJ1 in cells and show co-localization of antibody signal with the epitope tag signal .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate that specific binding is blocked.

• Cross-validation with multiple antibodies: Use antibodies raised against different epitopes of FTSJ1 to confirm consistent detection patterns.

• Mass spectrometry verification: Perform immunoprecipitation followed by mass spectrometry analysis to confirm that the antibody is pulling down FTSJ1 .

How should researchers interpret FTSJ1 expression levels in different experimental contexts?

When interpreting FTSJ1 expression data, consider these factors:

• Cancer prognosis correlation: High FTSJ1 expression in triple-negative breast cancer has been associated with poor prognosis. Use forest plot analysis to evaluate hazard ratios in different patient subgroups .

• Correlation with immune infiltration: Analyze the relationship between FTSJ1 expression and tumor-infiltrating immune cells using algorithms like ssGSEA. Higher FTSJ1 expression negatively correlates with CD8+ T cell infiltration in TNBC .

• Neurodevelopmental context: In neuronal cells, FTSJ1 expression levels may impact neurite morphology and function. Correlate expression levels with specific phenotypes like spine length and thickness .

• Tissue-specific expression patterns: Consider the normal tissue-specific expression patterns of FTSJ1 when interpreting changes in different experimental models or disease states.

• Interaction with WDR6: FTSJ1 functions in complex with WDR6, so co-expression levels of both partners should be considered when interpreting functional outcomes .

What statistical approaches are appropriate for analyzing FTSJ1 antibody-based experimental data?

Select appropriate statistical methods based on your experimental design:

• Survival analysis: For clinical correlations, use Kaplan-Meier survival analysis with log-rank tests to compare outcomes between high and low FTSJ1 expression groups. Report hazard ratios with confidence intervals .

• Comparative studies: For comparing FTSJ1 knockdown versus control conditions:

  • Use unpaired t-tests for comparing two groups when data follow normal distribution

  • For multiple group comparisons, use ANOVA with appropriate post-hoc tests

  • Present results as mean ± SEM for continuous variables

• Correlation analyses: When examining relationships between FTSJ1 expression and other variables (e.g., immune cell infiltration), use appropriate correlation coefficients (Pearson or Spearman) based on data distribution .

• Sample size considerations: Ensure adequate sample sizes by performing power calculations. For in vitro experiments, perform in triplicate and repeat at least three times to ensure reproducibility .

• Multiple testing correction: When analyzing large datasets (e.g., transcriptome analysis), apply appropriate corrections for multiple testing to control false discovery rates .

How can FTSJ1 antibodies be used in therapeutic development research?

FTSJ1 antibodies can facilitate therapeutic development through several approaches:

• Target validation: Use antibodies to confirm FTSJ1 knockdown or inhibition in preclinical studies evaluating FTSJ1 as a therapeutic target, particularly in triple-negative breast cancer .

• Drug screening: Employ antibodies in high-throughput screening assays to identify compounds that modulate FTSJ1 expression or activity. Molecular docking studies have identified potential FTSJ1 inhibitors including DAP, NV848, NV914, NV930, and PTC124 .

• Mechanism of action studies: Use antibodies to investigate how potential therapeutics affect FTSJ1-WDR6 complex formation or FTSJ1 interaction with its tRNA substrates .

• Biomarker development: Develop standardized FTSJ1 immunohistochemistry protocols for patient stratification in clinical trials, given its association with poor prognosis in TNBC .

• Combination therapy research: Investigate how FTSJ1 inhibition might synergize with immunotherapies, based on findings that FTSJ1 knockdown enhances tumor-infiltrating CD8+ T cells and increases sensitivity of TNBC to T cell killing .

What emerging technologies might enhance FTSJ1 antibody-based research?

Several cutting-edge technologies show promise for advancing FTSJ1 antibody-based research:

• Single-cell approaches: Apply single-cell proteomics and RNA sequencing to understand cell-to-cell variability in FTSJ1 expression and its impact on tRNA modification and translation.

• Proximity labeling techniques: Use BioID or APEX2 fused to FTSJ1 combined with antibody-based detection to identify novel interacting partners in different cellular compartments and conditions.

• Super-resolution microscopy: Apply techniques like STORM or PALM with FTSJ1 antibodies to precisely localize FTSJ1 within subcellular structures at nanometer resolution.

• In situ tRNA modification mapping: Develop methods combining FTSJ1 antibodies with techniques to visualize tRNA modifications in fixed cells to understand spatial regulation of tRNA modification.

• CRISPR screening with antibody readouts: Combine genome-wide CRISPR screens with high-content imaging using FTSJ1 antibodies to identify genes that regulate FTSJ1 expression, localization, or function.

• Computational modeling: Utilize structure-based approaches to model FTSJ1-WDR6-tRNA interactions and predict effects of mutations or inhibitors, subsequently validating predictions with antibody-based assays .