HKDC1 Antibody

What is HKDC1 and what are its primary biological functions?

HKDC1 (hexokinase domain containing 1) is a protein that catalyzes the phosphorylation of hexose to hexose 6-phosphate, albeit at lower efficiency compared to other hexokinases in the family. It demonstrates reduced glucose phosphorylating activity relative to other hexokinases but plays crucial roles in maintaining glucose homeostasis and hepatic lipid accumulation . Research indicates that HKDC1 is particularly important for maintaining whole-body glucose balance during pregnancy, although additional evidence is still required to fully confirm this specific role .

Beyond its metabolic functions, HKDC1 has been implicated in cancer progression mechanisms. Recent studies have revealed that HKDC1 promotes tumor immune evasion in hepatocellular carcinoma through a T cell-dependent manner by activating the STAT1/PD-L1 signaling pathway in tumor cells . This mechanism involves HKDC1 binding to and presenting cytosolic STAT1 to IFNGR1 on the plasma membrane following IFNγ-stimulation by associating with cytoskeleton protein ACTA2, resulting in STAT1 phosphorylation and nuclear translocation . Additionally, HKDC1's interaction with mitochondria has been identified as essential for its role in cancer progression, particularly in lung cancer .

What are the basic specifications of commercially available HKDC1 antibodies?

Commercial HKDC1 antibodies are available in various formats, with different specificities and applications. The most commonly used are polyclonal and monoclonal antibodies raised against specific regions of the HKDC1 protein. For example, the 25874-1-AP antibody is a rabbit polyclonal IgG that targets HKDC1 in applications including Western Blot, Immunohistochemistry, Immunofluorescence, Immunoprecipitation, and ELISA . It shows reactivity with human and mouse samples, with the immunogen being HKDC1 fusion protein Ag23045 .

Another example is the rabbit recombinant monoclonal HKDC1 antibody (EPR19835/ab209846), which is suitable for IP and WB applications with human and recombinant human samples . These antibodies are typically supplied in liquid form, with storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Proper storage conditions recommend keeping the antibodies at -20°C, where they remain stable for approximately one year after shipment . The antibodies recognize HKDC1 with a calculated molecular weight of 103 kDa (917 amino acids), though the observed molecular weight in experimental conditions can range from 82-90 kDa to 102 kDa .

Which sample types and species show reactivity with HKDC1 antibodies?

HKDC1 antibodies exhibit reactivity primarily with human and mouse samples, as confirmed by multiple validation studies. The 25874-1-AP antibody has demonstrated positive Western blot detection specifically in A549 cells (human lung carcinoma cell line) . For immunoprecipitation applications, this antibody has shown effectiveness with mouse skeletal muscle tissue . In immunohistochemistry applications, successful detection has been reported in both human and mouse skeletal muscle tissues .

The monoclonal antibody EPR19835 (ab209846) has been tested and validated in human samples, specifically showing clear detection in HeLa cells (human cervix adenocarcinoma epithelial cells), A549 cells, and human fetal kidney lysates . This antibody has also been demonstrated to successfully immunoprecipitate HKDC1 from HeLa cell lysates . Importantly, specificity testing through knockdown experiments confirms that these antibodies genuinely detect HKDC1 and not other hexokinases, as evidenced by the significant signal reduction in HKDC1-knockdown HeLa cells compared to cells with hexokinase I or hexokinase II knockdown . Cross-reactivity testing has shown that antibodies targeting specific hexokinase family members (like HK3) do not cross-react with HKDC1, confirming the specificity of hexokinase family antibodies .

What are the recommended protocols for Western blot applications with HKDC1 antibodies?

For optimal Western blot results with HKDC1 antibodies, researchers should follow these methodological steps based on validated protocols:

For the polyclonal antibody 25874-1-AP, the recommended dilution range is 1:200-1:1000 . More specifically, with the monoclonal antibody EPR19835, a 1:1000 dilution has been validated as effective . The blocking and dilution buffer should consist of 5% non-fat dry milk (NFDM) in TBST .

Sample preparation should include careful cell lysis to preserve the HKDC1 protein, which has a predicted molecular weight of 103 kDa but typically appears at approximately 102 kDa on Western blots . When analyzing cell lines, A549 and HeLa cells have been confirmed as positive controls that express detectable levels of HKDC1 . For tissue samples, human fetal kidney lysate has been validated as a positive control .

For secondary antibody detection, researchers can use goat anti-rabbit IgG H&L (HRP) at a 1:100,000 dilution for standard Western blot applications . The ECL (enhanced chemiluminescence) technique is recommended for development, with an optimal exposure time of approximately 3 minutes for clear visualization of the target band . To confirm antibody specificity, HKDC1-knockdown controls are essential, as they demonstrate significant signal reduction compared to wildtype samples and to cells with knockdown of other hexokinase family members .

How should immunohistochemistry protocols be optimized for HKDC1 detection?

For successful immunohistochemical detection of HKDC1 in tissue samples, specific protocol optimizations are necessary:

The recommended dilution range for the polyclonal antibody 25874-1-AP in IHC applications is 1:50-1:500 . Antigen retrieval is a critical step for optimal staining results. The suggested method involves using TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative method if needed .

Both human and mouse skeletal muscle tissues have been validated as positive controls for HKDC1 IHC staining . For formalin-fixed paraffin-embedded (FFPE) tissue sections, standard deparaffinization followed by the recommended antigen retrieval method is essential for antigen accessibility. When optimizing staining protocols, researchers should initially test a range of antibody concentrations within the recommended dilution range to determine the optimal concentration for their specific tissue samples.

Signal development should utilize an appropriate detection system compatible with rabbit primary antibodies. For challenging samples or weak signals, signal amplification methods may be considered, though validation against appropriate positive and negative controls is essential to confirm staining specificity. It's recommended that researchers titrate the antibody in each testing system to obtain optimal results, as the ideal concentration may be sample-dependent .

What are the key considerations for successful immunoprecipitation of HKDC1?

For effective immunoprecipitation of HKDC1, several methodological considerations are crucial:

The recommended antibody amount for the polyclonal antibody 25874-1-AP ranges from 0.5-4.0 μg for every 1.0-3.0 mg of total protein lysate . For the monoclonal antibody EPR19835, a dilution of 1:30 has been validated for successful immunoprecipitation from HeLa cell lysates .

Sample preparation is crucial for successful IP experiments. Mouse skeletal muscle tissue has been confirmed as a positive control for IP with the polyclonal antibody , while HeLa cell lysate works effectively with the monoclonal antibody . For detection of immunoprecipitated HKDC1, Western blot analysis can be performed using the same primary antibody at a dilution of 1:500 .

Importantly, specialized secondary antibodies that minimize detection of the IP antibody heavy and light chains are recommended for clearer results. For instance, VeriBlot for IP Detection Reagent (HRP) at 1:1000 dilution has been successfully used for detection following IP with EPR19835 . When analyzing results, researchers should be aware that while the predicted molecular weight of HKDC1 is 103 kDa, the observed band typically appears at approximately 102 kDa . Validation through appropriate controls is essential, comparing IP results with the specific HKDC1 antibody against a negative control using an isotype-matched antibody of irrelevant specificity .

How is HKDC1 expression altered in cancer tissues compared to normal tissues?

Research demonstrates significant alterations in HKDC1 expression in cancer tissues compared to normal counterparts. Transcriptomic data analysis from hepatocellular carcinoma (HCC) cases in The Cancer Genome Atlas (TCGA) has revealed substantially higher HKDC1 expression levels in clinical HCC lesion samples compared to matched non-cancerous tissues . This overexpression has been confirmed at the protein level through Western blot analysis, which demonstrated enhanced HKDC1 protein levels in HCC lesions compared to paired non-cancerous tissues .

Beyond hepatocellular carcinoma, elevated HKDC1 expression has been observed in other cancer types. For instance, qPCR results have shown that HKDC1 is expressed at substantially higher levels in tumor cells compared to other cell types . Cell line analyses confirm detectable expression in multiple cancer cell lines, including A549 (lung carcinoma), HeLa (cervical adenocarcinoma), and HepG2 (hepatocellular carcinoma) .

Importantly, the clinical significance of HKDC1 overexpression has been demonstrated by its negative correlation with progression-free survival in HCC patients treated with atezolizumab (anti-PD-L1 therapy) . This suggests HKDC1 expression levels may serve as a potential biomarker for predicting treatment response to immunotherapy. Interestingly, analysis of publicly available CCLE RNAi and CRISPR gene dependency data indicates that HKDC1 expression does not have an obvious relationship with viability in HCC cell lines, suggesting its role in cancer progression might be mediated through other mechanisms beyond direct effects on cancer cell proliferation .

What is HKDC1's role in tumor immune evasion mechanisms?

HKDC1 plays a significant role in tumor immune evasion, particularly in hepatocellular carcinoma, through multiple mechanistic pathways. Research has demonstrated that HKDC1 promotes tumor immune evasion in a T cell-dependent manner by activating the STAT1/PD-L1 signaling pathway in tumor cells . This occurs through a specific molecular mechanism where HKDC1 binds to and presents cytosolic STAT1 to IFNGR1 on the plasma membrane following IFNγ-stimulation, accomplished via association with cytoskeleton protein ACTA2, which results in STAT1 phosphorylation and subsequent nuclear translocation .

Experimental evidence from multiple models supports HKDC1's immunosuppressive role. In mouse models, HKDC1 knockout significantly suppressed HCC incidence and development compared to wild-type mice . Flow cytometric analysis revealed that the proportion of exhausted CD8+ T cells (characterized by PD-1 and LAG-3 expression) was significantly lower in HKDC1 knockout mice than in wild-type counterparts . Similarly, tumor growth was suppressed in mice inoculated with HKDC1 knockdown cancer cells, with the infiltrated CD8+ T cells exhibiting lower exhaustion markers (PD-1 and LAG-3) and higher effector molecule expression (IFNγ and GzmB) compared to control mice .

In vitro CD8+ T cell killing assays further demonstrated that HKDC1 knockdown could sensitize cancer cells to cytolysis by CD8+ T cells . Flow cytometric analysis showed that CD8+ T cells co-cultured with HKDC1 knockdown cancer cells displayed lower exhaustion markers but higher effector molecule expression, indicating that tumor cell-mediated immune suppression was alleviated by HKDC1 depletion . Importantly, transcriptomic data from clinical HCC samples in TCGA indicated that HKDC1 expression level positively correlated with CD8+ T cell exhaustion, further supporting its role in promoting tumor immune evasion .

How does HKDC1 inhibition affect response to immune checkpoint blockade therapy?

Inhibiting HKDC1 demonstrates significant potential for enhancing the efficacy of immune checkpoint blockade (ICB) therapy. Research has shown that HKDC1 inhibition in combination with anti-PD-1/PD-L1 therapy enhances in vivo T cell antitumor responses in liver cancer models in male mice . This combinatorial approach represents a promising strategy to overcome resistance to immunotherapy.

The mechanism behind this synergistic effect appears to be related to HKDC1's role in the STAT1/PD-L1 signaling pathway. By inhibiting HKDC1, the subsequent activation of STAT1 and upregulation of PD-L1 on tumor cells is reduced, potentially making tumor cells more susceptible to immune detection and elimination . Clinical sample analysis indicates a significant correlation among HKDC1 expression, STAT1 phosphorylation, and survival outcomes in patients with hepatocellular carcinoma treated with atezolizumab (anti-PD-L1) .

These findings suggest that HKDC1 expression levels could potentially serve as a biomarker for predicting response to ICB therapy, with higher HKDC1 expression being associated with poorer treatment outcomes. The negative correlation between elevated HKDC1 expression and progression-free survival in HCC patients treated with atezolizumab supports this hypothesis . By targeting HKDC1 in combination with ICB therapy, researchers may be able to enhance antitumor immune responses and potentially overcome resistance mechanisms, presenting a novel therapeutic strategy for cancer treatment .

What experimental controls should be used to validate HKDC1 antibody specificity?

To ensure robust validation of HKDC1 antibody specificity, researchers should implement a comprehensive set of experimental controls:

Genetic manipulation controls are essential for definitive validation. HKDC1 knockdown or knockout samples provide the most stringent specificity controls, as demonstrated in the literature where HKDC1-knockdown HeLa cell lysates showed significant signal reduction compared to wild-type cells . Importantly, controls should also include cells with knockdown of other hexokinase family members (HK1, HK2) to confirm that the antibody does not cross-react with related proteins .

Positive control samples with confirmed HKDC1 expression are crucial for validating detection sensitivity. A549 cells, HeLa cells, and human fetal kidney lysates have been experimentally validated as reliable positive controls . For tissue-based applications, human and mouse skeletal muscle tissues have been confirmed as appropriate positive controls .

Cross-reactivity testing should be performed using recombinant protein panels including multiple hexokinase family members. Dot blot analysis with His-tagged human HK1, HK2, HK3, and HKDC1 fragments can confirm antibody specificity within the hexokinase family . This approach has demonstrated that antibodies targeting specific hexokinases (e.g., HK3) do not cross-react with HKDC1, highlighting the importance of thorough cross-reactivity testing .

For immunohistochemistry applications, peptide competition assays can provide additional specificity validation. By pre-incubating the antibody with excess immunizing peptide, specific staining should be blocked or significantly reduced. Additionally, comparative analysis across multiple HKDC1 antibodies targeting different epitopes can provide convergent validation of specificity when similar staining patterns are observed.

How can researchers optimize HKDC1 antibody detection in challenging samples?

Optimizing HKDC1 antibody detection in challenging samples requires strategic methodological adjustments:

For samples with low HKDC1 expression, signal enhancement techniques can be employed. These include using more sensitive detection systems such as enhanced chemiluminescence (ECL) with extended exposure times of approximately 3 minutes, as validated in published protocols . For immunohistochemistry applications in tissues with low expression or high background, antigen retrieval optimization is critical, with TE buffer at pH 9.0 recommended as the primary method, though citrate buffer at pH 6.0 can serve as an effective alternative .

Sample preparation optimization is essential for maintaining protein integrity and enhancing detection sensitivity. For protein extraction, using buffers containing protease inhibitors helps prevent degradation of HKDC1, which has a calculated molecular weight of 103 kDa . When working with tissue samples, fresh or properly preserved specimens yield better results than extensively processed or archived samples.

Antibody concentration and incubation conditions require careful titration for each specific application and sample type. For Western blot applications, the recommended dilution range of 1:200-1:1000 should be tested to determine optimal concentration . For immunohistochemistry, a broader range of 1:50-1:500 is suggested, with the optimal dilution being sample-dependent . Extended primary antibody incubation at 4°C overnight rather than shorter incubations at room temperature can enhance specific binding in challenging samples.

For samples with high background or non-specific binding, blocking optimization is crucial. Using 5% non-fat dry milk (NFDM) in TBST has been validated as an effective blocking solution for Western blot applications . Additionally, including appropriate washing steps with optimized duration and buffer composition helps reduce background signal while maintaining specific detection of the target protein.

What is the significance of HKDC1's mitochondrial interaction in experimental design?

HKDC1's interaction with mitochondria represents a critical consideration for experimental design in cancer research. Evidence indicates that this interaction is essential for HKDC1's role in cancer progression, particularly in lung cancer, and without this interaction, mitochondrial function is compromised . This knowledge should inform how researchers design and interpret HKDC1-focused experiments.

When studying HKDC1 function in cancer models, subcellular localization analysis should be incorporated to assess mitochondrial association. Techniques such as subcellular fractionation followed by Western blot analysis, or co-immunoprecipitation studies targeting mitochondrial proteins can reveal this critical interaction. Confocal microscopy with appropriate mitochondrial markers (such as MitoTracker dyes) and HKDC1 immunofluorescence can provide visual confirmation of co-localization.

For functional studies, researchers should consider designing experiments that specifically disrupt or enhance the HKDC1-mitochondria interaction, rather than simply modulating total HKDC1 expression. This could involve creating targeted mutations in HKDC1 domains responsible for mitochondrial binding without affecting catalytic activity, or vice versa. Comparing the effects of these specific disruptions against total HKDC1 knockdown would provide insights into which HKDC1 functions are mitochondria-dependent versus independent.

When interpreting metabolic phenotypes resulting from HKDC1 manipulation, researchers should consider dual effects on both glycolytic activity and mitochondrial function. Comprehensive metabolic analysis should include measurements of both glycolytic parameters (glucose uptake, lactate production) and mitochondrial function indicators (oxygen consumption rate, ATP production, mitochondrial membrane potential). This integrated approach would provide a more complete understanding of how HKDC1 influences cellular energetics through both direct catalytic activity and mitochondrial interactions .

How can HKDC1 expression analysis contribute to patient stratification for immunotherapy?

HKDC1 expression analysis shows significant potential for improving patient stratification in immunotherapy, particularly for hepatocellular carcinoma. Research has demonstrated that elevated HKDC1 expression is significantly negatively correlated with progression-free survival in HCC patients treated with atezolizumab (anti-PD-L1) . This finding suggests that HKDC1 expression levels could serve as a predictive biomarker for response to immune checkpoint blockade therapy.

To implement HKDC1 expression analysis in clinical settings, researchers should establish standardized protocols for quantitative assessment. This could involve immunohistochemical staining of tumor biopsies using validated HKDC1 antibodies with defined scoring systems to categorize expression levels. The polyclonal antibody 25874-1-AP at a dilution of 1:50-1:500 has been validated for IHC applications and could be standardized for clinical use . Alternatively, qPCR-based analysis of HKDC1 mRNA expression in tumor samples could provide quantitative data for patient stratification.

Integrated biomarker approaches combining HKDC1 expression with other immune markers might provide enhanced predictive power. Since HKDC1 influences tumor immune evasion through modulation of CD8+ T cell function and STAT1/PD-L1 signaling , combining HKDC1 expression analysis with assessment of tumor-infiltrating lymphocytes, PD-L1 expression, and STAT1 phosphorylation status could create a more comprehensive predictive signature. Clinical studies have already indicated a correlation among HKDC1 expression, STAT1 phosphorylation, and survival in patients with hepatocellular carcinoma treated with atezolizumab , supporting the potential of this integrated approach.

For practical implementation, researchers should develop clinically validated cutoff values for HKDC1 expression that can reliably separate potential responders from non-responders to immunotherapy. This would require retrospective analysis of HKDC1 expression in larger cohorts of immunotherapy-treated patients with documented response data, followed by prospective validation in clinical trials specifically designed to test HKDC1-guided treatment selection.

What novel therapeutic approaches target the HKDC1-STAT1-PD-L1 axis?

Emerging therapeutic approaches targeting the HKDC1-STAT1-PD-L1 axis represent promising strategies for enhancing cancer immunotherapy. Research has demonstrated that HKDC1 inhibition in combination with anti-PD-1/PD-L1 therapy enhances in vivo T cell antitumor responses in liver cancer models . This combinatorial approach could overcome resistance mechanisms to immune checkpoint blockade therapy.

Direct HKDC1 inhibition strategies could involve the development of small molecule inhibitors specifically targeting HKDC1 enzymatic activity or its protein-protein interactions. Since HKDC1 binds to and presents cytosolic STAT1 to IFNGR1 following IFNγ-stimulation by associating with cytoskeleton protein ACTA2 , compounds disrupting these specific interactions could potentially block the downstream STAT1 phosphorylation and nuclear translocation that leads to PD-L1 upregulation.

RNA interference-based therapeutics represent another approach for targeting HKDC1. Experimental evidence has shown that HKDC1 knockdown can sensitize cancer cells to cytolysis by CD8+ T cells and reduce expression of exhaustion markers on tumor-infiltrating T cells . Delivering siRNA or shRNA targeting HKDC1 to tumor cells, potentially using nanoparticle-based delivery systems, could achieve similar effects in clinical settings. The CRISPR-Cas9 system has already been successfully employed to create HKDC1 knockout cell lines and animal models for research purposes , demonstrating the feasibility of genetic targeting approaches.

Disrupting HKDC1's interaction with mitochondria presents another therapeutic avenue, as this interaction has been shown to be essential for HKDC1's role in cancer progression . Compounds that specifically interfere with this interaction without affecting other HKDC1 functions could potentially provide targeted anti-cancer effects with reduced impact on normal glucose metabolism. Any therapeutic approach would benefit from combination with existing immune checkpoint inhibitors, as the evidence suggests a synergistic effect when HKDC1 inhibition is combined with anti-PD-1/PD-L1 therapy .

What are the key considerations for integrating HKDC1 research into broader cancer immunology studies?

Integrating HKDC1 research into broader cancer immunology studies requires several strategic considerations to maximize impact and translational relevance. Researchers should prioritize evaluation of HKDC1 expression across diverse cancer types beyond hepatocellular carcinoma, where its role has been most extensively characterized . This expanded analysis would determine whether HKDC1's role in immune evasion represents a common mechanism across multiple cancers or is context-specific.

Methodologically, research designs should incorporate immune component analysis alongside HKDC1 manipulation. When conducting HKDC1 knockdown or overexpression studies, comprehensive immune profiling of the tumor microenvironment should be performed, including assessment of T cell subsets, exhaustion marker expression, and effector molecule production . The validated applications of HKDC1 antibodies in Western blot, immunohistochemistry, and immunoprecipitation provide valuable tools for these integrated analyses .

Mechanistic investigations should expand beyond the established HKDC1-STAT1-PD-L1 axis to explore potential interactions with other immune signaling pathways. Since HKDC1 serves as a critical link between metabolism and immune regulation, studies examining how metabolic alterations resulting from HKDC1 modulation impact immune cell function would be particularly valuable. The unique role of HKDC1 in connecting cytoskeleton with STAT1 activation suggests it may participate in other immunomodulatory processes involving cytoskeletal rearrangements.

For translational relevance, predictive biomarker development should combine HKDC1 expression with established immune markers. The correlation between HKDC1 expression and CD8+ T cell exhaustion in clinical samples suggests that integrated biomarker panels incorporating both HKDC1 and T cell functionality markers could provide enhanced predictive power for immunotherapy response. As therapeutic development progresses, researchers should focus on strategies that combine HKDC1 targeting with existing immunotherapies, since evidence suggests this combinatorial approach enhances antitumor immune responses .