C11orf73 Antibody

Basic Research Questions

• What is C11orf73/HIKESHI and what experimental systems are suitable for studying its function?

  C11orf73, also known as HIKESHI (meaning "to put out fire" in Japanese), functions as a specific nuclear import carrier for HSP70 proteins following heat-shock stress. It mediates the nucleoporin-dependent translocation of ATP-bound HSP70 proteins into the nucleus, which is essential for protecting cells from heat shock damage .

  For experimental systems, oligodendroglial cell lines (such as FBD-102b) have been used to study C11orf73 mutations, while gastric cancer cell lines have been employed to investigate its role in cancer progression . COS-7 cells are suitable for biochemical experiments requiring high protein expression levels, with transfection efficiencies reaching approximately 75% compared to 25-27% in FBD-102b cells .

• What applications are C11orf73 antibodies validated for and what are their typical reactivity profiles?

  C11orf73 antibodies have been validated for multiple applications including:

  • Western Blotting (WB)

  • Flow Cytometry (FACS)

  • Immunohistochemistry on paraffin-embedded sections (IHC-p)

  • Immunofluorescence (IF)

  • ELISA

  Regarding reactivity, commercially available antibodies show cross-reactivity patterns with multiple species:

  Antibody Catalog	Reactivity	Applications	Host
  ABIN654735	Human, Hamster	WB, FACS, IHC (p)	Rabbit
  ABIN6745127	Human, Mouse, Rat, Hamster, Horse, Cow, Dog, Pig, Bat, Monkey	WB	Rabbit
  ABIN6750374	Human, Mouse, Rat, Horse	WB	Rabbit
  ABIN7165631	Human	ELISA, IHC	Rabbit

  Most antibodies demonstrate at least human reactivity, while some offer broader species coverage .

• What epitope regions of C11orf73 are typically targeted by antibodies and how does this affect experimental design?

  C11orf73 antibodies target various epitope regions including:

  • N-terminal region (AA 6-35)

  • Mid-region (AA 37-86)

  • C-terminal region

  • Full-length protein (AA 1-197)

  When designing experiments, researchers should consider that the N-terminal region contains the Cys4 residue that, when mutated to Ser (C4S), is associated with infantile hypomyelinating leukoencephalopathy. Antibodies targeting this region may show differential binding to wild-type versus mutant proteins . For studies examining C11orf73 mutations, antibodies targeting different epitopes should be compared, as the C4S mutation has been shown to decrease protein expression levels while still maintaining detectable amounts in nervous tissues .

Advanced Research Questions

• How can researchers effectively detect C11orf73 mutant proteins and their subcellular localization?

  To study C11orf73 mutant proteins (particularly the pathological C4S and V54L variants), researchers should implement a multi-faceted approach:

  1. Dual immunostaining strategy: Use both anti-C11ORF73 antibodies and tag-specific antibodies (such as anti-GFP for GFP-tagged constructs) to confirm similar localization patterns .

  2. Subcellular fractionation: Isolate lysosomal fractions using buffer containing HEPES-NaOH, NaCl, MgCl₂, EDTA, and protease inhibitors, followed by immunoprecipitation with lysosome-specific markers .

  3. Co-localization studies: Employ antibodies against lysosomal markers (LAMP1, Cathepsin D) and potential binding partners (Filamin A) for immunofluorescence microscopy to confirm mutant protein aggregation in lysosomes .

  Studies have shown that C4S mutant proteins, unlike wild-type C11orf73, localize and aggregate in lysosomes and specifically interact with Filamin A, which anchors transmembrane proteins to the actin cytoskeleton .

• What are the methodological considerations for studying the role of HIKESHI in heat shock response?

  When investigating HIKESHI's role in heat shock response, researchers should consider:

  1. Temperature control protocols: Studies on gastric cancer cells have shown HIKESHI function is activated specifically during heat shock conditions, not at normal temperatures. Establish precise temperature controls (typically 42-43°C for heat shock experiments) .

  2. HSP70 nuclear translocation assays: Monitor HSP70 localization using immunofluorescence with anti-HSP70 antibodies before and after heat shock, in the presence or absence of HIKESHI .

  3. siRNA knockdown optimization: HIKESHI suppression using siRNA shows no effect on cell viability at normal temperatures but inhibits HSP70 nuclear transport and suppresses cell viability during heat shock. Optimize transfection conditions to achieve maximal knockdown without off-target effects .

  4. ATP-dependent binding studies: Since HIKESHI specifically translocates ATP-bound HSP70 (not ADP-bound forms), design experiments to distinguish between these states using appropriate nucleotide analogs or ATP-depletion strategies .

• How can researchers effectively purify and identify C11orf73-binding proteins?

  For purification and identification of C11orf73 binding partners:

  1. Sequential affinity chromatography: Use tandem-tagged (FLAG and hexa-histidine) C11orf73 constructs expressed in appropriate cell lines (e.g., human glial T98G cells), with sequential purification through M2-conjugated and Ni-NTA agarose columns .

  2. Sample preparation for mass spectrometry:

     • Concentrate purified fractions using 20% TCA precipitation

     • Digest with Trypsin Gold (1 U/mL overnight)

     • Prevent disulfide bond reformation by treating with iodoacetamide

     • Separate trypsin-digested peptides by gradient chromatography (0-60% acetonitrile in 0.1M trifluoroacetic acid)

  3. Co-immunoprecipitation validation: Confirm identified interactions using standard immunoprecipitation with anti-C11ORF73 or anti-binding partner antibodies (e.g., anti-Filamin A) followed by immunoblotting .

  This approach has successfully identified Filamin A as a specific binding partner of the C4S mutant but not wild-type C11orf73 .

• What are the considerations for using C11orf73/HIKESHI antibodies in cancer research?

  When utilizing C11orf73/HIKESHI antibodies in cancer research:

  1. Tissue-specific expression analysis: HIKESHI expression in gastric cancer tissues has been associated with lymphatic invasion progression. Use immunohistochemistry with optimized antibody dilutions for tissue microarrays and paraffin sections .

  2. Correlation with clinical parameters: Design studies to correlate HIKESHI expression (by immunoblotting or immunohistochemistry) with patient outcomes, cancer stage, and treatment response, particularly in hyperthermia-based therapies .

  3. Functional studies: Combine HIKESHI knockdown with heat shock treatment to assess potential therapeutic approaches. Research has shown that suppressing HIKESHI during heat shock (but not at normal temperatures) inhibits HSP70 nuclear transport and suppresses cancer cell viability .

  4. Antibody selection for cancer tissues: For cancer tissue studies, select antibodies that have been specifically validated in the cancer type of interest, as expression patterns may differ across tissue types .

• How do plasmid construction approaches differ for studying wild-type versus mutant C11orf73?

  When designing experiments to compare wild-type and mutant C11orf73:

  1. Gene amplification and vector selection: Amplify human full-length c11orf73 from brain cDNAs using SuperScript III reverse transcriptase and ligate with appropriate vectors (e.g., pEGFP-C1 for GFP-fusion proteins) .

  2. Site-directed mutagenesis: Generate the disease-associated mutations (C4S: 11G-to-C; V54L: 160G-to-C) using site-directed mutagenesis kits with the wild-type construct as template .

  3. Transfection optimization: Different cell lines require different transfection reagents and protocols. For FBD-102b cells, ScreenFect A or ScreenFect A Plus have been used with medium replacement four hours post-transfection .

  4. Stable cell line generation: For long-term studies, generate stable cell lines by selecting transfected cells with G418 (0.1250 mg/mL) for approximately 14 days, then collect resistant colonies for phenotypic comparison .

  5. Viability assessment: Confirm cell viability using trypan blue exclusion to ensure that attached trypan-blue-incorporating cells comprise less than 5% of all cells in culture .

• What antibody validation steps are critical for ensuring specificity in C11orf73/HIKESHI studies?

  To ensure antibody specificity for C11orf73/HIKESHI:

  1. Positive and negative tissue controls: Test antibodies on tissues known to express HIKESHI positively and negatively before experimental use .

  2. Western blot validation: Confirm specific binding at the expected molecular weight (approximately 21.6 kDa for human HIKESHI) .

  3. Knockdown/knockout validation: Compare antibody signals in wild-type cells versus those with siRNA-mediated knockdown or genetic knockout of HIKESHI .

  4. Cross-reactivity assessment: Validate species cross-reactivity claims by testing on samples from multiple species if cross-species comparisons are planned .

  5. Secondary antibody controls: Include controls with secondary antibodies alone to identify potential non-specific binding. For fluorescent detection systems (like Odyssey Infrared Imager), optimize secondary antibody dilutions (typically 1:10,000 for IRDye-conjugated antibodies) .

• How can researchers effectively study the relationship between C11orf73/HIKESHI and the mTOR signaling pathway?

  To investigate the relationship between C11orf73/HIKESHI and mTOR signaling:

  1. Lysate preparation for signaling analysis: Prepare cell lysates from wild-type and C11orf73 mutant-expressing cells using appropriate buffers with phosphatase inhibitors (Na₃VO₄, NaF) to preserve phosphorylation states .

  2. Phosphorylation status assessment: Use phospho-specific antibodies against mTOR pathway components (S6, 4E-BP1) in immunoblotting to assess how C11orf73 mutations affect lysosomal mTOR signaling .

  3. Inhibitor studies: Employ mTOR inhibitors (e.g., rapamycin) to determine whether phenotypes associated with C11orf73 mutations are dependent on mTOR signaling .

  4. Morphological differentiation assays: Compare morphological differentiation between parental cells, wild-type C11orf73-expressing cells, and C4S mutant-expressing cells to connect signaling alterations with functional outcomes .

  Research has shown that cells expressing C4S mutant proteins exhibit decreased lysosomal mTOR signaling, including reduced S6 and 4E-BP1 phosphorylation, which is essential for oligodendrocyte differentiation and myelination .