DECR1 Antibody

What is the cellular localization of DECR1 and how can it be visualized?

DECR1 primarily localizes to the mitochondria, which has been confirmed through both immunocytochemistry and Western blot analysis of subcellular fractions. For proper visualization, researchers should use:

• Immunocytochemistry with appropriate mitochondrial co-staining: DECR1 can be visualized using specific antibodies coupled with Alexa Fluor 488 secondary antibody, while mitochondria can be labeled using MitoTracker Red and nuclei with DAPI .

• Subcellular fractionation: When performing Western blot analysis of DECR1 expression, proper separation of cytosolic, mitochondrial, and nuclear fractions should be confirmed using specific markers such as cytochrome-C for mitochondria and PARP for nuclear fractions .

How is DECR1 expression regulated in different tissue types?

DECR1 expression varies significantly across tissue types and is influenced by several factors:

• Hormone regulation: DECR1 is a negatively-regulated androgen receptor (AR) target gene. It is downregulated by dihydrotestosterone (DHT) and synthetic androgens like R1881, while its expression increases in response to androgen receptor antagonists like enzalutamide (ENZ) and androgen deprivation therapy (ADT) .

• Tissue-specific expression: DECR1 shows highest expression in cardiomyocytes compared to other cardiac cell types .

• Pathological conditions: Expression is significantly upregulated in prostate cancer tissues compared to benign prostate tissues, with higher expression correlating with increased Gleason score and advanced disease stage . Similarly, DECR1 levels are elevated in diabetic cardiomyopathy models .

How can DECR1 antibodies be utilized to investigate its role in cancer progression and treatment resistance?

DECR1 antibodies serve as crucial tools for exploring the relationship between DECR1 and cancer progression:

• Tissue microarrays: Quantitative immunohistochemistry (IHC) using DECR1 antibodies can determine expression differences between malignant and benign tissues, with studies showing significantly increased expression in malignant regions compared to benign ones within the same core .

• Therapy response biomarker: DECR1 expression increases following androgen deprivation therapy and AR-targeted therapies like enzalutamide, making DECR1 antibodies valuable for monitoring treatment response and potential resistance mechanisms. This can be evaluated through Western blotting and IHC of patient-derived explants (PDEs) treated with AR antagonists .

• Metastasis studies: Since DECR1 knockdown suppresses tumor cell proliferation and metastasis, antibodies can track DECR1 expression in metastatic models and patient samples to assess correlation with disease progression .

What experimental approaches can be used to investigate DECR1's role in mitochondrial dysfunction and oxidative stress?

Several sophisticated approaches utilizing DECR1 antibodies can elucidate its role in mitochondrial function:

• Mitochondrial ROS detection: Combine DECR1 immunostaining with MitoSOX Red to correlate DECR1 expression with mitochondrial reactive oxygen species generation, particularly in conditions of metabolic stress such as high glucose/high palmitate exposure .

• Mitochondrial membrane potential analysis: Use JC-1 dye in conjunction with DECR1 antibody labeling to assess the relationship between DECR1 expression and mitochondrial permeability transition, a critical step in apoptosis induction .

• Protein-protein interaction studies: Co-immunoprecipitation with DECR1 antibodies can identify interaction partners such as PDK4, which may mediate downstream effects on mitochondrial function and fatty acid oxidation .

What are the optimal conditions for using DECR1 antibodies in immunohistochemistry of clinical samples?

For reliable IHC results with DECR1 antibodies in clinical samples:

• Sample preparation: Formalin-fixed, paraffin-embedded (FFPE) tissues should be sectioned at 4-5 μm thickness and undergo appropriate antigen retrieval.

• Antibody validation: Always validate antibody specificity using positive controls (tissues known to express DECR1 highly, such as prostate cancer or cardiac tissue) and negative controls (antibody diluent without primary antibody) .

• Quantification methods: Implement digital pathology approaches for quantitative analysis of DECR1 expression. This is particularly important when comparing expression between malignant and benign regions within the same tissue sample .

• Signal amplification: Consider using polymer-based detection systems for enhanced sensitivity, especially when examining subtle differences in expression levels between experimental groups .

How can researchers effectively analyze DECR1 expression changes in response to experimental interventions?

To accurately measure changes in DECR1 expression:

• mRNA quantification: Use qRT-PCR with appropriate reference genes (such as GUSB and L19) for normalization. Calculate relative expression using the comparative CT method, setting control/vehicle-treated samples to one .

• Protein quantification: For Western blot analysis, normalize DECR1 expression to appropriate loading controls such as HSP90. Perform densitometry quantification for accurate comparison between experimental conditions .

• Time-course experiments: When studying regulation by hormones or other factors, include multiple time points to capture both acute and chronic effects on DECR1 expression .

How should researchers address discrepancies between DECR1 mRNA and protein expression data?

When faced with discrepancies between mRNA and protein expression:

• Post-translational modifications: Consider investigating potential modifications that might affect protein stability or detection. Use phosphatase or deglycosylation treatments before Western blotting if appropriate.

• Antibody epitope accessibility: Ensure the antibody's epitope isn't masked by protein interactions or conformational changes under your experimental conditions.

• Half-life differences: Measure protein stability using cycloheximide chase experiments to determine if differences arise from altered protein turnover rather than transcriptional regulation .

• Cell-type specific effects: As observed in cardiac tissue, DECR1 mRNA may be elevated only in specific cell types (cardiomyocytes) but not in others (cardiac fibroblasts and endothelial cells) . Consider cell isolation techniques to examine expression in specific cell populations.

What are the critical considerations when designing DECR1 knockdown or overexpression experiments?

For effective genetic manipulation of DECR1:

• Knockdown verification: Always verify knockdown efficiency at both mRNA (qRT-PCR) and protein levels (Western blot) before interpreting functional outcomes .

• Proper controls: Include both negative controls (non-targeting siRNA/scramble constructs) and positive controls (genes with known phenotypes in your system) alongside DECR1 manipulation .

• Rescue experiments: To confirm specificity of observed effects, perform rescue experiments by reintroducing DECR1 expression in knockdown models or by countering DECR1 overexpression with specific inhibitors .

• Phenotypic assessments: Select appropriate functional readouts based on the pathway being studied. For mitochondrial function, combine assessments of ROS production, membrane potential, and morphological changes. For cancer models, assess proliferation, migration, and in vivo tumor growth .

How can DECR1 antibodies be utilized to investigate its role in ferroptosis and lipid peroxidation?

DECR1's involvement in ferroptosis offers exciting research opportunities:

• Lipid peroxidation detection: Combine DECR1 immunostaining with lipid peroxidation markers to investigate the relationship between DECR1 expression and ferroptosis induction in cancer cells .

• Polyunsaturated fatty acid (PUFA) metabolism: Use DECR1 antibodies in conjunction with lipidomic analyses to correlate DECR1 expression with changes in PUFA profiles and oxidation products .

• Therapeutic resistance mechanisms: Investigate whether DECR1-mediated ferroptosis resistance contributes to treatment failure in cancer by examining DECR1 expression in resistant versus sensitive cell populations .

What experimental approaches can elucidate the therapeutic potential of targeting DECR1 in cardiac and cancer contexts?

To explore DECR1 as a therapeutic target:

• Small molecule screening: Use DECR1 antibodies to verify target engagement of potential inhibitors like Atranorin and Kurarinon, which have shown promise in ameliorating DCM through binding to and inhibiting DECR1 .

• Patient-derived models: Apply DECR1 antibodies in patient-derived explants (PDEs) and organoids to assess the efficacy of DECR1 targeting across diverse patient backgrounds and genetic contexts .

• Combination therapies: Investigate synergistic effects of DECR1 inhibition with standard-of-care treatments in both cardiac and cancer contexts, using DECR1 antibodies to confirm target modulation .