DEPP1 Antibody

What is DEPP1 and what are its basic structural characteristics?

DEPP1 (Decidual protein induced by progesterone 1) is a protein with a canonical length of 212 amino acid residues and a molecular mass of approximately 23.4 kDa in humans. The protein contains a conserved PTS2 (peroxisomal targeting sequence type 2) nested within an N-terminal t-snare domain, which contributes to its subcellular localization and function . DEPP1 is also known by several synonyms including DEPP, FIG, Fseg, protein DEPP1, DEPP1 autophagy regulator, and C10orf10 . The protein has been identified in several species including mouse, rat, bovine, and chimpanzee, indicating evolutionary conservation .

Where is DEPP1 expressed and what is its subcellular localization?

DEPP1 is expressed in various tissues, including pancreas, placenta, ovary, testis, and kidney . At the subcellular level, DEPP1 localizes primarily to mitochondria and the cytoplasm . Recent research has specifically confirmed that upon expression, DEPP1 localizes inside mitochondria, which is critical for its function in promoting autophagy in cardiomyocytes . This mitochondrial localization is essential for understanding its role in organelle homeostasis during conditions such as hypoxia and ischemia.

What are the primary functions of DEPP1 in cellular processes?

DEPP1 functions as a critical modulator of FOXO3-induced autophagy via increased cellular reactive oxygen species (ROS) . Recent research has revealed that DEPP1 is a key component in the cardiac remodeling that occurs with chronic ischemia . It is both necessary and sufficient for hypoxia-induced autophagy and triglyceride accumulation in cardiomyocytes . DEPP1 appears to play a specific role in promoting the loss of mitochondria and peroxisomes through autophagy in response to HIF activation, but interestingly not in response to other autophagy inducers such as mTOR inhibition .

What are the most effective applications for DEPP1 antibodies in research?

DEPP1 antibodies are primarily used for immunodetection of the protein in various experimental setups. The most common applications include ELISA, Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blot (WB), and Immunocytochemistry (ICC) . Each application provides different insights: ELISA allows for quantitative analysis of DEPP1 levels, Western blotting helps determine protein expression and molecular weight, while immunofluorescence and immunohistochemistry help visualize the subcellular localization and tissue distribution of DEPP1 . When selecting an antibody, researchers should consider the specific validation data for their intended application as performance can vary significantly.

How should researchers optimize immunofluorescence protocols for DEPP1 detection in cardiomyocytes?

For optimal immunofluorescence detection of DEPP1 in cardiomyocytes, researchers should consider several key protocol modifications. First, fixation methods are critical - 4% paraformaldehyde for 10-15 minutes typically preserves DEPP1 structure while maintaining cellular architecture. Given DEPP1's mitochondrial localization, co-staining with mitochondrial markers (such as TOM20 or MitoTracker) is recommended for colocalization studies . Permeabilization should be gentle (0.1-0.2% Triton X-100) to maintain mitochondrial integrity while allowing antibody access. For primary antibody incubation, overnight incubation at 4°C generally yields better signal-to-noise ratios than shorter incubations. When analyzing results, confocal microscopy is preferable to standard fluorescence microscopy to accurately resolve mitochondrial localization patterns.

What considerations are important when selecting DEPP1 antibodies for experimental use?

When selecting DEPP1 antibodies, researchers should consider several critical factors beyond just specificity. First, evaluate the antibody's validation in your specific application (WB, IF, IHC, ELISA) and target species, as cross-reactivity profiles vary significantly between antibodies . Second, consider the epitope location—antibodies targeting different regions of DEPP1 may perform differently depending on protein conformation and interactions. Third, for colocalization studies with mitochondrial markers, fluorophore selection is crucial to prevent spectral overlap. Fourth, examine literature citations to verify the antibody's performance in similar experimental contexts . Finally, for quantitative applications, consider whether unconjugated or directly conjugated (HRP, FITC) antibodies are more appropriate for your specific detection system and sensitivity requirements.

How can researchers effectively design experiments to study DEPP1 induction under hypoxic conditions?

To effectively study DEPP1 induction under hypoxic conditions, researchers should implement a multi-faceted experimental approach. First, establish a time-course study exposing cells (preferably cardiomyocytes or other DEPP1-expressing cell types) to varying oxygen concentrations (1%, 2%, 5%) for different durations (6, 12, 24, 48 hours) . Include the HIF stabilizer FG-4592 as a positive control group to confirm HIF-dependency . For protein analysis, perform Western blotting with validated DEPP1 antibodies alongside HIF1α detection to correlate expression patterns. Complement protein studies with RT-qPCR to measure DEPP1 mRNA induction, designing primers spanning exon junctions to ensure specificity. To confirm direct HIF regulation, perform chromatin immunoprecipitation (ChIP) assays targeting the conserved hypoxia response element (HRE) upstream of the DEPP1 transcriptional start site . Finally, include ARNT knockout controls to verify the HIF-dependency of DEPP1 induction under hypoxia.

What are the methodological approaches for studying DEPP1's role in mitochondrial and peroxisomal autophagy?

To investigate DEPP1's role in mitochondrial and peroxisomal autophagy, researchers should employ several complementary methodological approaches. First, establish DEPP1 knockout and overexpression models using CRISPR-Cas9 gene editing or lentiviral transduction in relevant cell types such as cardiomyocytes . For mitochondrial autophagy (mitophagy) assessment, implement both static and flux measurements using mitochondrial markers (TOM20, COXIV) combined with autophagy markers (LC3B, p62) . Fluorescence microscopy with mt-Keima or mt-mCherry-GFP tandem reporters can track mitophagy flux in real-time. For peroxisomal autophagy (pexophagy), monitor peroxisomal markers (PEX14, catalase) and analyze colocalization with autophagosomes. Electron microscopy provides ultrastructural validation of organelle engulfment by autophagosomes. To connect DEPP1 function with these processes, perform rescue experiments by reintroducing wild-type DEPP1 or mutants lacking specific domains (e.g., the t-snare domain) into knockout cells, then quantify the restoration of autophagy phenotypes .

How can researchers effectively validate the specificity of DEPP1 antibodies for their experimental systems?

Validating DEPP1 antibody specificity requires a systematic approach combining multiple techniques. First, perform Western blot analysis using positive control samples (tissues known to express DEPP1 such as kidney or pancreas) alongside negative controls . Critical validation should include lysates from DEPP1 knockout cells generated via CRISPR-Cas9, which should show complete absence of the specific band . For further validation, compare the reactivity pattern of multiple antibodies targeting different DEPP1 epitopes—consistent detection patterns increase confidence in specificity. Peptide competition assays where the antibody is pre-incubated with excess immunizing peptide should abolish specific signals. For immunofluorescence applications, parallel staining with multiple validated antibodies and correlation with GFP-tagged DEPP1 in overexpression systems provides additional confirmation. Finally, siRNA knockdown of DEPP1 should result in proportional signal reduction across all validation techniques.

What is the role of DEPP1 in ischemic cardiomyopathy and how can DEPP1 antibodies help investigate this?

DEPP1 has been identified as a key component in cardiac remodeling during chronic ischemia . Recent research has revealed that DEPP1 is induced under hypoxia in a HIF-dependent manner and localizes inside mitochondria, where it promotes autophagy of mitochondria and peroxisomes . This process contributes to the pathophysiology of ischemic cardiomyopathy, as DEPP1 is both necessary and sufficient for hypoxia-induced autophagy and triglyceride accumulation in cardiomyocytes . Notably, DEPP1 loss increases cardiomyocyte survival during chronic HIF activation and decreases cardiac dysfunction in mouse hearts with chronic HIF activation caused by VHL loss . DEPP1 antibodies are crucial tools for investigating these processes, enabling the detection and quantification of DEPP1 expression in ischemic tissues, visualization of its subcellular localization via immunofluorescence, and assessment of its induction kinetics in response to hypoxia through Western blotting and immunohistochemistry .

How does DEPP1 interact with the HIF pathway, and what experimental approaches can elucidate this relationship?

DEPP1 interacts with the HIF pathway through a multi-faceted relationship that can be investigated through several experimental approaches. Research has established that DEPP1 is a direct transcriptional target of HIF, containing a conserved hypoxia response element (HRE) upstream of its transcriptional start site . Chromatin immunoprecipitation (ChIP) assays have confirmed that HIF1α binds to this HRE during hypoxia in an ARNT-dependent manner . Interestingly, DEPP1 and HIF appear to participate in a positive feedback loop, as DEPP1 loss dampens the induction of HIF1α and other HIF-responsive genes under hypoxia . To investigate this relationship, researchers can employ CRISPR-Cas9 editing of the DEPP1 HRE, which abrogates hypoxia-induced DEPP1 protein abundance . Time-course experiments monitoring both HIF1α stabilization and DEPP1 induction can help establish the temporal relationship between these events. Co-immunoprecipitation studies may reveal whether DEPP1 physically interacts with HIF pathway components. Additionally, comparative transcriptomics between wild-type and DEPP1-knockout cells under hypoxia can identify the broader impact of DEPP1 on HIF-regulated gene networks.

What is the significance of DEPP1's role in autophagy regulation, and how can this be studied in different pathological contexts?

DEPP1's role in autophagy regulation represents a critical link between hypoxic signaling and organelle homeostasis with significant implications for multiple pathological conditions. DEPP1 functions as a modulator of FOXO3-induced autophagy via increased cellular ROS and is necessary for mitochondrial and peroxisomal autophagy specifically in response to HIF activation . This selective role distinguishes DEPP1-mediated autophagy from other autophagy induction pathways, such as those triggered by mTOR inhibition . To study this in different pathological contexts, researchers should implement a multi-model approach combining in vitro, ex vivo, and in vivo systems. Cell-type specific inducible DEPP1 knockout models can reveal tissue-specific roles in autophagy regulation. Autophagic flux should be assessed using tandem fluorescent reporters (mRFP-GFP-LC3 or organelle-specific variants) in disease models such as ischemia-reperfusion injury, neurodegenerative conditions, or cancer hypoxia models. Therapeutic modulation of DEPP1 expression or function (using inducible systems or pharmaceutical approaches) can help determine whether intervention in this pathway affects disease progression. Comparative studies of DEPP1-dependent versus independent autophagy pathways will elucidate the unique contribution of DEPP1 to pathological processes across different disease contexts.

What are common technical challenges when using DEPP1 antibodies and how can they be overcome?

Researchers frequently encounter several technical challenges when using DEPP1 antibodies. First, background staining is common, particularly in immunohistochemistry and immunofluorescence applications. This can be minimized by implementing more stringent blocking protocols (extending blocking time to 2 hours with 5-10% serum plus 1% BSA) and titrating antibody concentrations precisely . Second, inconsistent detection between batches can occur, which necessitates validation of each new lot against a known positive control. Third, mitochondrial localization of DEPP1 can complicate accurate signal detection; optimize permeabilization conditions carefully to ensure antibody access while preserving mitochondrial integrity . Fourth, some commercially available antibodies show cross-reactivity with similarly sized proteins; confirm specificity using DEPP1 knockout controls generated by CRISPR-Cas9 . Finally, low signal-to-noise ratios in Western blotting applications can be improved by using signal enhancement systems with low-fluorescence PVDF membranes for enhanced detection of mitochondrial proteins.

How can researchers distinguish between true DEPP1 signals and non-specific antibody binding in their experiments?

Distinguishing true DEPP1 signals from non-specific binding requires implementation of multiple rigorous controls. The gold standard approach involves parallel analysis of samples from DEPP1 knockout models generated via CRISPR-Cas9, which should exhibit complete absence of specific signals . Secondary-only controls are essential to identify background from detection systems. Peptide competition assays, where the primary antibody is pre-incubated with excess immunizing peptide, should significantly diminish specific signals while leaving non-specific binding unaffected. For Western blotting, analysis of molecular weight is crucial—DEPP1 shows a characteristic band at approximately 23.4 kDa . In immunofluorescence applications, compare staining patterns from multiple antibodies targeting different DEPP1 epitopes; overlapping signals strongly suggest true detection. Correlation of staining intensity with DEPP1 expression levels in physiologically relevant conditions (such as hypoxia-induced upregulation) provides functional validation . Finally, immunoprecipitation followed by mass spectrometry can definitively identify DEPP1 in antibody-captured complexes, particularly valuable for validating novel antibodies.

What methods are effective for quantifying DEPP1 expression levels in tissue samples and how should data be normalized?

Accurate quantification of DEPP1 expression in tissue samples requires careful consideration of methodology and normalization strategies. For protein quantification, Western blotting with fluorescent secondary antibodies provides superior linear dynamic range compared to chemiluminescence detection. ELISA methods offer greater sensitivity and throughput but require extensively validated antibody pairs . For immunohistochemistry quantification, digital image analysis with machine learning algorithms that distinguish subcellular compartments can separate mitochondrial from cytoplasmic DEPP1 signals . When normalizing Western blot data, traditional housekeeping proteins may be inappropriate in hypoxic conditions where many "stable" proteins change; consider using total protein normalization methods such as Ponceau S or Stain-Free technology. For RT-qPCR quantification of DEPP1 mRNA, normalization should employ multiple reference genes validated for stability under experimental conditions, especially important given HIF's broad transcriptional effects. When comparing samples across different pathological states, stratified normalization approaches may be necessary—normalizing mitochondrial DEPP1 to mitochondrial markers (TOMM20) and cytosolic DEPP1 to cytosolic markers separately provides more accurate assessment of compartment-specific changes.

How can DEPP1 antibodies be utilized in multi-parameter flow cytometry for analyzing autophagy in heterogeneous cell populations?

Multi-parameter flow cytometry with DEPP1 antibodies enables sophisticated analysis of autophagy in heterogeneous cell populations, particularly valuable for analyzing primary tissue samples. This approach requires careful optimization of a fixation and permeabilization protocol that preserves both surface markers for cell type identification and allows antibody access to intracellular DEPP1, preferably using a saponin-based permeabilization system . Researchers should combine DEPP1 antibodies (preferably conjugated to bright fluorophores like PE or APC) with established autophagy markers (LC3-II, p62) and organelle-specific markers (mitochondrial: TOMM20; peroxisomal: PEX14) . Cell type-specific markers should be included to distinguish subpopulations (e.g., CD45+ leukocytes, CD31+ endothelial cells, cardiac troponin T+ cardiomyocytes). For functional assessment, include ROS indicators such as CellROX, given DEPP1's role in ROS-mediated autophagy . This comprehensive panel allows correlation of DEPP1 expression levels with autophagy activation status across different cell types within the same sample, enabling identification of cell populations particularly sensitive to DEPP1-mediated autophagy during pathological conditions.

What are the considerations for using DEPP1 antibodies in high-content screening approaches to identify modulators of mitochondrial autophagy?

High-content screening (HCS) with DEPP1 antibodies presents a powerful approach for identifying modulators of mitochondrial autophagy, but requires careful optimization of several parameters. First, select cell types with robust DEPP1 expression and clear mitochondrial localization; primary cardiomyocytes or cell lines with confirmed DEPP1 expression patterns are ideal . Optimize a fixation protocol that preserves mitochondrial morphology while allowing antibody penetration, typically using 2-4% paraformaldehyde with a controlled permeabilization period. The immunostaining protocol should include DEPP1 antibodies alongside mitochondrial markers (TOMM20), autophagy markers (LC3B), and nuclear counterstains to enable automated cell segmentation . For screening design, implement positive controls including hypoxia exposure or HIF stabilizers (FG-4592) to induce DEPP1 expression . Image analysis algorithms should quantify multiple parameters: DEPP1 expression levels, mitochondrial mass, degree of DEPP1-mitochondria colocalization, autophagosome formation, and mitochondria-autophagosome colocalization. This multi-parametric approach allows identification of compounds that specifically modulate DEPP1-dependent mitophagy while distinguishing them from general autophagy modulators.

How can researchers develop in vivo imaging approaches to monitor DEPP1 activity in disease models?

Developing in vivo imaging approaches to monitor DEPP1 activity in disease models requires sophisticated strategies that bridge molecular biology and imaging technology. The most promising approach involves generating transgenic reporter animals expressing a DEPP1-fluorescent protein fusion under control of the endogenous DEPP1 promoter, ideally using CRISPR knock-in techniques to maintain physiological regulation . For cardiac applications, implement mouse models with tissue-specific expression of the hypoxia response element from the DEPP1 gene driving a luciferase reporter, enabling bioluminescence imaging of HIF-induced DEPP1 transcription during ischemia . For more detailed cellular resolution, develop a split-fluorescent protein complementation system where one fragment is fused to DEPP1 and the complementary fragment to mitochondrial or autophagosomal markers, generating signal only when DEPP1 localizes to these structures . In established disease models like ischemic cardiomyopathy, intravital microscopy through cardiac imaging windows can track fluorescent reporters in real-time. For clinical translation, develop DEPP1-specific PET tracers based on radiolabeled antibody fragments (e.g., nanobodies) that can penetrate tissues and bind to DEPP1 with high specificity, potentially enabling monitoring of DEPP1 induction in human cardiovascular diseases.