RSPH14 Antibody

What is RSPH14 and what cellular functions does it regulate?

RSPH14 (Radial Spoke Head 14 Homolog) is a protein encoded by the RSPH14 gene, also previously known as RTDR1 (Rhabdoid Tumor Deletion Region Gene 1). Recent research has demonstrated that RSPH14 plays significant roles in cellular proliferation, migration, invasion, and apoptosis pathways. In particular, RSPH14 appears to modulate these cellular functions by influencing the expression of key signaling proteins, including RelA (NF-κBp65), CDH2, and AKT1 . These interactions suggest RSPH14 participates in cellular signaling networks critical for both normal cell function and pathological processes.

In which cancer types has RSPH14 expression been investigated?

Current research has primarily investigated RSPH14 expression in hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). In both cancer types, RSPH14 has been found to be upregulated compared to adjacent normal tissues . Additionally, database analyses suggest RSPH14 may serve as a prognostic marker in renal cancer, indicating its potential relevance across multiple cancer types . The expression pattern of RSPH14 in other malignancies remains an active area of investigation.

What evidence supports RSPH14 as a potential therapeutic target in cancer?

Several lines of evidence support RSPH14 as a potential therapeutic target:

What are the commercially available RSPH14 antibodies and their recommended applications?

Commercially available RSPH14 antibodies include polyclonal antibodies suitable for various experimental applications. For instance, biotin-conjugated RSPH14 polyclonal antibodies raised in rabbit against human RSPH14 (UniProt ID: Q9UHP6) are specifically designed for ELISA applications . These antibodies target recombinant RSPH14 protein (amino acids 1-230) and are purified using Protein G, with purity exceeding 95% . For immunohistochemistry applications, primary rabbit anti-human RSPH14 antibodies (such as PA5-113475, 1:200 dilution, Invitrogen Inc.) have been successfully employed in research settings .

How does RSPH14 knockdown affect cancer cell phenotypes at the molecular level?

RSPH14 knockdown elicits significant changes in cancer cell phenotypes through modulation of multiple molecular pathways:

• Proliferation inhibition: Knockdown of RSPH14 in HCC cell lines (BEL-7404 and SMMC-7721) significantly reduces cell counts in BrdU assays, decreases cell proliferation in Celigo image cytometry, and diminishes colony formation ability (P < 0.05)

• Apoptosis induction: Flow cytometry analyses reveal that RSPH14 depletion significantly increases the percentage of apoptotic cells compared to control groups (P < 0.05)

• Migration and invasion suppression: RSPH14 knockdown significantly inhibits cell migration and invasion capabilities as measured by scratch wound-healing and Transwell assays

• Molecular signaling changes: Western blot analyses show that RSPH14 knockdown alters the expression of 14 proteins involved in tumorigenesis-associated signaling pathways, apoptosis, and epithelial-mesenchymal transition (EMT). Most notably, RSPH14 depletion downregulates RelA (NF-κBp65), CDH2, and AKT1

• RelA-mediated effects: Functional rescue experiments demonstrate that RelA overexpression can attenuate the inhibitory effects of RSPH14 knockdown on cell proliferation and migration, indicating that RSPH14 exerts its oncogenic functions at least partially through RelA-dependent mechanisms

What are the optimal methodological approaches for RSPH14 detection in tissue samples?

For optimal RSPH14 detection in tissue samples, immunohistochemistry protocols have been validated with the following methodology:

• Sample preparation: Tissue specimens should be embedded in paraffin and sectioned at 4 mm thickness, followed by deparaffinization and rehydration procedures

• Antibody selection and dilution: Primary rabbit anti-human RSPH14 antibody (such as PA5-113475, Invitrogen) should be used at a 1:200 dilution. Secondary antibody (goat anti-rabbit IgG) should be applied at a 1:200 dilution

• Incubation conditions: Primary antibody incubation should be performed overnight at 4°C, followed by secondary antibody incubation for 60 minutes at room temperature

• Visualization: 3,3′-Diaminobenzidine should be used as the chromogen, with hematoxylin counterstaining for nuclei visualization

• Imaging: Images should be obtained using an optical microscope equipped with a digital camera

• Scoring system: RSPH14 expression can be scored based on the percentage of positive tumor cells: 0 (<15%), 1+ (16–30%), 2+ (31–60%), and 3+ (61–100%)

• Evaluation protocol: All stained sections should be independently evaluated by at least two investigators to ensure scoring reliability and reproducibility

How do RSPH14 expression patterns differ between cancer subtypes and what are the implications for prognosis?

RSPH14 expression patterns vary significantly between cancer subtypes, with important prognostic implications:

RSPH14 expression increases with tumor progression parameters in HCC, including:

• Higher expression with increased tumor differentiation degree

• Higher expression with lymph node metastasis

• Higher expression with advanced tumor stage

All these associations were statistically significant (P < 0.05) .

These expression patterns suggest that RSPH14 may serve as a valuable prognostic biomarker across multiple cancer types, though additional research is needed to fully characterize its utility in cancers beyond HCC.

What are the challenges in developing effective anti-RSPH14 therapeutic strategies?

Developing effective anti-RSPH14 therapeutic strategies presents several significant challenges:

• Target specificity: Ensuring therapeutic agents specifically target RSPH14 without affecting related proteins or pathways requires extensive validation studies

• Delivery mechanisms: Determining optimal delivery methods to achieve sufficient knockdown or inhibition of RSPH14 in tumor tissues while minimizing off-target effects

• Resistance mechanisms: Understanding potential compensatory pathways that might emerge following RSPH14 inhibition, particularly given its interactions with multiple signaling proteins (RelA, CDH2, AKT1)

• Patient stratification: Identifying patient subpopulations most likely to benefit from RSPH14-targeted therapies based on expression levels and molecular profiles

• Combination approaches: Determining which existing therapies might synergize with RSPH14 inhibition to maximize anti-tumor effects

• Biomarker development: Establishing reliable biomarkers to monitor therapeutic efficacy and predict patient response

The research to date suggests targeting the RSPH14-RelA axis may be particularly promising, as RelA overexpression partially rescues the phenotypic effects of RSPH14 knockdown .

What are the optimal conditions for using RSPH14 antibodies in different experimental protocols?

The optimal conditions for using RSPH14 antibodies vary by experimental protocol:

Immunohistochemistry (IHC):

• Primary antibody: Rabbit anti-human RSPH14 (PA5-113475, Invitrogen)

• Dilution: 1:200

• Incubation: Overnight at 4°C

• Secondary antibody: Goat anti-rabbit IgG at 1:200 dilution for 60 minutes at room temperature

• Chromogen: 3,3′-diaminobenzidine with hematoxylin counterstaining

Enzyme-Linked Immunosorbent Assay (ELISA):

• Recommended antibody: Biotin-conjugated polyclonal RSPH14 antibody

• Storage: -20°C or -80°C, avoiding repeated freeze-thaw cycles

• Buffer composition: 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative

Western Blotting:
While specific conditions for Western blotting were not explicitly detailed in the provided references, this technique was successfully employed to confirm RSPH14 knockdown efficiency in lentiviral-transfected HCC cells . When adapting protocols, researchers should optimize antibody dilutions, incubation times, and blocking conditions based on manufacturer recommendations and preliminary testing.

How can researchers effectively validate RSPH14 knockdown or overexpression models?

Effective validation of RSPH14 knockdown or overexpression models requires a multi-faceted approach:

• Transcript-level validation:

  • qRT-PCR to quantify RSPH14 mRNA levels

  • Comparison with appropriate housekeeping genes

  • Statistical analysis to confirm significant differences between experimental and control groups

• Protein-level validation:

  • Western blotting using validated anti-RSPH14 antibodies

  • Densitometric analysis normalized to loading controls

  • Statistical comparison between experimental and control conditions

• Functional validation:

  • Proliferation assays (BrdU, MTT, colony formation)

  • Apoptosis assessment (flow cytometry)

  • Migration and invasion assays (scratch wound-healing, Transwell)

  • Documenting phenotypic changes consistent with expected outcomes of modulation

• In vivo validation (for comprehensive models):

  • Xenograft mouse models to assess tumor growth characteristics

  • Immunohistochemical analysis of tumor tissues

  • Measurement of tumor volume, weight, and other relevant parameters

• Downstream target validation:

  • Assessment of known downstream effectors (e.g., RelA, CDH2, AKT1)

  • Rescue experiments to confirm specificity of observed effects

What considerations should researchers take into account when interpreting RSPH14 immunohistochemistry results?

When interpreting RSPH14 immunohistochemistry results, researchers should consider:

• Scoring methodology: Use a standardized scoring system based on percentage of positive tumor cells (0: <15%; 1+: 16–30%; 2+: 31–60%; 3+: 61–100%)

• Inter-observer variability: Involve at least two independent investigators to evaluate stained sections, with procedures for resolving discrepancies through discussion

• Tissue heterogeneity: Account for potential heterogeneity within tumor samples by examining multiple fields and regions

• Controls: Include appropriate positive and negative controls to validate staining specificity

• Technical variables: Consider fixation time, antigen retrieval methods, and antibody lot variations that might affect staining intensity

• Correlation with clinical data: Interpret RSPH14 expression in conjunction with clinical parameters (tumor stage, differentiation, patient outcomes)

• Subcellular localization: Note subcellular distribution patterns of RSPH14 staining, as localization may provide additional functional insights

• Comparative analysis: Compare expression between tumor and adjacent normal tissues from the same patient when possible to establish relative expression levels

How can researchers design experiments to elucidate the molecular mechanisms by which RSPH14 influences cancer progression?

To elucidate the molecular mechanisms by which RSPH14 influences cancer progression, researchers should design experiments that:

• Establish protein-protein interaction networks:

  • Co-immunoprecipitation to identify RSPH14 binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry to identify novel interaction partners

• Analyze signaling pathway modulation:

  • Phosphoproteomic analysis following RSPH14 modulation

  • Western blotting for key signaling molecules (e.g., RelA, AKT1)

  • Pathway-specific reporter assays (e.g., NF-κB reporters)

• Perform transcriptomic analyses:

  • RNA sequencing following RSPH14 knockdown or overexpression

  • Pathway enrichment analysis to identify affected biological processes

  • Validation of key differentially expressed genes

• Conduct rescue experiments:

  • Overexpression of downstream effectors (e.g., RelA) in RSPH14-knockdown cells

  • Assessment of whether phenotypic changes are rescued

  • Quantification of rescue efficiency

• Utilize domain-specific mutations:

  • Creation of RSPH14 mutants lacking specific functional domains

  • Evaluation of which domains are critical for observed phenotypes

  • Assessment of whether specific mutations affect protein-protein interactions

• Employ pharmacological approaches:

  • Use of specific inhibitors of potential downstream pathways

  • Determination of whether inhibitors phenocopy RSPH14 knockdown effects

  • Combination studies to identify synergistic interactions

• Analyze epigenetic regulation:

  • Investigation of whether RSPH14 influences gene expression through epigenetic mechanisms

  • Chromatin immunoprecipitation to identify potential DNA binding sites

  • Analysis of histone modifications at RSPH14-regulated genes