GOT1 Antibody

What is GOT1 and what is its primary function in cellular metabolism?

GOT1 (glutamic-oxaloacetic transaminase 1, soluble) is a cytoplasmic enzyme that catalyzes the reversible reaction of L-aspartate and alpha-ketoglutarate into oxaloacetate and L-glutamate. This enzyme plays a critical role in carbon and nitrogen metabolism within cells. GOT1 serves as a key component of the malate-aspartate shuttle system, which is essential for maintaining cellular redox balance by facilitating the transfer of reducing equivalents between the cytosol and mitochondria. Additionally, GOT1 can potentially control intracellular levels of reactive oxygen species (ROS) through its involvement in NADPH synthesis, thereby contributing to cellular redox homeostasis . The enzyme has a calculated molecular weight of 46 kDa, though it is typically observed at 43-46 kDa in experimental settings .

What applications are recommended for GOT1 antibodies in research?

GOT1 antibodies have been validated for multiple research applications as shown in the following table:

Researchers should note that dilution optimization is essential for each experimental system, as optimal concentrations may vary depending on sample type and preparation methods .

How should researchers validate the specificity of GOT1 antibodies?

To ensure reliable results, researchers should implement a multi-step validation approach:

• Knockout/knockdown validation: Testing antibody specificity using GOT1 knockout or knockdown systems is the gold standard. The search results indicate successful validation using CRISPR/Cas9-mediated knockout and doxycycline-inducible shRNA knockdown systems targeting GOT1 .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (43-46 kDa for GOT1) .

• Positive control tissues: Include known GOT1-expressing samples such as HepG2 cells, L02 cells, or brain tissue from mouse or rat models .

• Cross-reactivity assessment: Test the antibody against samples from multiple species if working with non-human models, as GOT1 antibodies show reactivity with human, mouse, and rat samples .

• Blocking peptide experiments: If available, use the immunogen peptide to competitively inhibit antibody binding and confirm specificity.

What are the best practices for using GOT1 antibodies in studies of cancer metabolism?

When investigating GOT1's role in cancer metabolism, researchers should consider several methodological approaches:

• Paired analysis of expression and metabolites: Combine GOT1 protein detection (via western blot or immunohistochemistry) with metabolomic analysis of related metabolites such as aspartate, glutamate, and oxaloacetate. In GOT1 knockdown experiments, aspartate levels have been successfully used as a biochemical readout of GOT1 inhibition .

• Cell line selection: Include both GOT1-sensitive and GOT1-insensitive cancer cell lines for comparative analysis. Studies have demonstrated variability in GOT1 dependency across pancreatic cancer cell lines, with 12 of 18 tested lines showing significant sensitivity to GOT1 knockdown .

• Normal tissue controls: Include non-transformed cell lines as controls, such as human pancreatic stellate cells (hPSC), human lung fibroblasts (IMR-90), or human non-transformed pancreatic exocrine cells (hPNE), which have shown minimal sensitivity to GOT1 inhibition .

• Functional pathway analysis: Combine GOT1 detection with analysis of related metabolic pathways, including glutamine metabolism, redox balance mechanisms, and ferroptosis sensitivity markers.

• In vivo validation: Confirm in vitro findings using xenograft tumor models with inducible GOT1 knockdown systems, monitoring both tumor growth kinetics and molecular markers like Ki-67 for proliferation .

How can researchers effectively use GOT1 antibodies to investigate ferroptosis mechanisms?

Investigating GOT1's role in ferroptosis requires a methodical approach:

• Combinatorial experimental design: Test GOT1 inhibition in combination with known ferroptosis inducers such as RSL3 (a GPX4 inhibitor), erastin (a system x_c^- inhibitor), or BSO (a glutathione synthesis inhibitor) .

• Lipid peroxidation assessment: Use lipid peroxidation sensors such as C11-BODIPY to measure the accumulation of lipid reactive oxygen species following GOT1 inhibition alone or in combination with ferroptosis inducers .

• Rescue experiments: Include ferroptosis inhibitors like ferrostatin-1 (Fer-1) to confirm the mechanism of cell death .

• Labile iron detection: Implement image-based detection of labile iron using fluorescent probes such as RhoNox-1, as described in the research protocol: "Cells were treated with Hoechst (1 μg/mL) and RhoNox-1 (Goryo, GC901) at 500 nM for 6 h then imaged using a Cytation5 Cell Imaging Multi-Mode Reader" .

• Temporal analysis: Monitor changes over time, as GOT1 knockdown has been shown to induce G1 cell cycle arrest after 5 days of treatment, indicating that timing is critical for observing specific phenotypes .

How does GOT1 contribute to metabolic reprogramming in cancer cells?

GOT1 plays several sophisticated roles in cancer metabolic reprogramming:

• Non-canonical glutamine metabolism: In pancreatic ductal adenocarcinoma (PDA), GOT1 participates in a non-canonical glutamine metabolism pathway where glutamine-derived aspartate is converted by GOT1 into oxaloacetate, which is then converted to malate and pyruvate, increasing the NADPH/NADP+ ratio to maintain redox homeostasis .

• Redox balance maintenance: GOT1 contributes to maintaining cellular redox balance through its involvement in NADPH production, which is essential for detoxifying reactive oxygen species (ROS). This function becomes particularly critical under oxidative stress conditions common in cancer microenvironments .

• Ferroptosis resistance: GOT1 activity appears to protect cancer cells from ferroptosis, as GOT1 suppression potentiates sensitivity to ferroptosis inducers. This suggests GOT1 may function as a metabolic vulnerability that can be exploited for cancer treatment .

• Cell cycle regulation: GOT1 inhibition leads to G1 cell cycle arrest in sensitive cancer cell lines, indicating a role in supporting cancer cell proliferation beyond its classical metabolic functions .

• Interaction with oncogenic signaling: GOT1 expression is regulated by specific oncogenes and transcription factors, suggesting integration between metabolic reprogramming and oncogenic signaling pathways .

What methodologies can researchers use to study the role of GOT1 in immune cell function?

Recent studies have highlighted GOT1's importance in immune cell metabolism, particularly in T cells. Researchers can investigate this using:

• Conditional knockout models: Generate immune cell-specific GOT1 knockout models to examine its function in specific immune cell populations.

• Functional immune assays: Assess T cell proliferation, cytokine production, and cytotoxic activity in the context of GOT1 manipulation. Studies have shown that GOT1 silencing led to reduced percentage and number of antigen-specific CD8+ T cells responding to infection and decreased IFN-γ production .

• Metabolic flux analysis: Use isotope-labeled metabolites (particularly glutamine) to trace GOT1-dependent metabolic pathways in immune cells.

• Redox status assessment: Measure NAD+/NADH ratios and ROS levels, as GOT1 has been shown to maintain NAD+/NADH ratios in T cells, which is critical for effector T cell formation .

• Transcriptional and epigenetic analysis: Examine how GOT1 deficiency influences transcriptional profiles and epigenetic landscapes of immune cells, particularly focusing on genes involved in demethylation (e.g., Kdm6b and Tet1) .

• In vivo infection models: Utilize infection models like lymphocytic choriomeningitis virus to study GOT1's role in acute immune responses .

What factors might affect GOT1 antibody performance in experimental conditions?

Several factors can influence antibody performance that researchers should consider:

• Sample preparation method: Different lysis buffers may affect epitope accessibility. For GOT1, which is involved in metabolic functions, preserving native protein structure may be important for certain applications.

• Fixation protocols: For immunofluorescence studies, the fixation method can significantly impact antibody binding. Paraformaldehyde fixation at 4% is commonly used, but optimization might be necessary for specific tissues.

• Expression level variations: GOT1 expression levels vary across tissue types and disease states, potentially requiring different antibody dilutions for optimal detection.

• Post-translational modifications: GOT1 function may be regulated by post-translational modifications, which could affect antibody recognition depending on the epitope location.

• Storage conditions: GOT1 antibodies should be stored according to manufacturer recommendations, typically at -20°C in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and are generally stable for one year after shipment .

How should researchers interpret differences in GOT1 dependency across cancer cell lines?

Studies have shown variable dependency on GOT1 across cancer cell lines, particularly in pancreatic cancer. When interpreting these differences:

• Metabolic profiling: Perform comprehensive metabolic profiling to identify alternative metabolic pathways that might compensate for GOT1 inhibition in resistant cells.

• Genetic background analysis: Consider the genetic background of different cell lines, as certain oncogenic drivers may influence GOT1 dependency.

• Microenvironmental factors: Evaluate how nutrient availability in the microenvironment might affect GOT1 dependency. Studies have shown that dietary factors can influence nutrient composition within pancreatic tumors, affecting sensitivity to metabolic interventions .

• Temporal dynamics: Assess both immediate and long-term responses to GOT1 inhibition, as some cells may initially respond but later develop adaptive resistance.

• Combinatorial approach: Test combinations of GOT1 inhibition with other metabolic inhibitors, as some studies have shown that inhibition of glutathione biosynthesis (using BSO) in combination with GOT1 inhibition enhances therapeutic efficacy .

What are promising directions for developing GOT1-targeted cancer therapies?

Based on current research, several promising therapeutic approaches are emerging:

• Small molecule inhibitors: Multiple drug discovery campaigns are underway to develop small molecule inhibitors of GOT1, though further optimization is needed for in vivo studies .

• Combination therapies: Combining GOT1 inhibition with inducers of ferroptosis (such as erastin, RSL3, or BSO) shows promise for enhanced anti-tumor effects through synergistic mechanisms .

• Dietary interventions: Exploiting metabolic vulnerabilities through dietary manipulation of cystine availability might provide a complementary approach to pharmacological GOT1 inhibition .

• Biomarker-guided therapy: Identifying biomarkers of GOT1 dependency could help stratify patients who might benefit most from GOT1-targeted therapies.

• Immune modulation: Understanding GOT1's role in T cell metabolism opens possibilities for combining GOT1 inhibition with immunotherapy approaches in appropriate contexts .

How can researchers assess the potential off-target effects of GOT1 inhibition?

When evaluating GOT1 as a therapeutic target, researchers should systematically assess potential off-target effects:

• Non-transformed cell toxicity: Include non-transformed control cells (such as hPSC, IMR-90, and hPNE) in cytotoxicity assays, as they have shown minimal sensitivity to GOT1 inhibition, suggesting a potential therapeutic window .

• Immune function assessment: Given GOT1's role in T cell function, evaluate how GOT1 inhibition affects anti-tumor immune responses, particularly CD8+ T cell expansion and function .

• Metabolic bypass mechanisms: Identify potential metabolic bypass mechanisms that might develop in response to chronic GOT1 inhibition, as cells have shown plasticity in adapting to metabolic perturbations.

• Tissue-specific effects: Assess GOT1 dependency across different tissues to anticipate potential organ-specific toxicities, particularly in metabolically active tissues.

• Reversibility testing: Determine whether the effects of GOT1 inhibition are reversible, as studies have shown that cells can regain proliferative capacity upon removal of genetic GOT1 inhibition .