DHRSX Antibody

What is DHRSX and why is it significant in research?

DHRSX (also known as CXorf11, DHRS5X, or DHRSXY) is a 330 amino acid protein belonging to the short-chain dehydrogenase/reductase (SDR) superfamily. It functions as an oxidoreductase containing coenzyme binding and substrate binding sites, suggesting roles in cellular metabolism . Research has identified DHRSX as a novel non-classical secretory protein that plays a positive regulatory role in starvation-induced autophagy. Its high conservation across species (humans, mice, rats, cows, dogs, chickens, fruit flies, zebrafish, and mosquitoes) suggests fundamental biological importance . The gene encoding DHRSX is uniquely positioned in the pseudoautosomal region 1 (PAR1) of both X and Y chromosomes .

What experimental applications are validated for DHRSX antibodies?

DHRSX antibodies have been validated for multiple research applications with specific performance parameters:

These applications enable comprehensive investigation of DHRSX expression, localization, and interactions in various experimental systems .

What is the tissue expression profile of DHRSX?

DHRSX exhibits a wide expression pattern across human tissues with particularly high expression in the pancreas. Interestingly, expression levels in cell lines are notably decreased compared to primary tissues. This differential expression pattern suggests potential tissue-specific functions and regulation .

How does DHRSX influence autophagy and what experimental designs best capture this activity?

DHRSX has been identified as a positive regulator of starvation-induced autophagy. Experimental evidence demonstrates that:

To effectively study DHRSX's role in autophagy, researchers should design experiments that include:

• Autophagy induction using Earle's balanced salt solution (EBSS)

• Late-stage autophagy inhibition using chloroquine (CQ) to assess autophagic flux

• Monitoring of multiple autophagy markers (LC3-II, p62, polyQ80)

• Analysis of AKT/mTOR phosphorylation and Beclin1 expression

• Appropriate controls for each experimental condition

What mechanisms underlie DHRSX's non-classical secretion and how can they be experimentally verified?

Despite bioinformatic predictions suggesting a classical secretion pathway (SignalP software predicted a signal peptide cleavage site between amino acids 31-32), experimental evidence confirms DHRSX as a non-classical secretory protein:

• N-terminal sequencing revealed that secreted DHRSX retains its complete predicted signal peptide sequence

• DHRSX secretion is not suppressed by Bafilomycin A1 (Baf.A1), a classical secretion inhibitor

• The observed molecular weight of secreted DHRSX (~38 kDa) more closely matches its full-length form (36.4 kDa) than the predicted form with cleaved signal peptide (33 kDa)

To verify these properties, researchers should:

• Purify secreted DHRSX from cell supernatants using affinity chromatography

• Perform N-terminal sequencing of the purified protein

• Compare secretion levels with and without classical secretion inhibitors

• Analyze molecular weight patterns via SDS-PAGE and Western blotting

How does DHRSX interact with the AKT/mTOR/Beclin1 pathway in autophagy regulation?

DHRSX appears to modulate autophagy through the AKT/mTOR/Beclin1 signaling axis. When DHRSX is overexpressed in starvation-induced U2OS cells:

Conversely, knockdown of endogenous DHRSX results in decreased Beclin1 protein levels. Notably, these effects are primarily observed under starvation conditions, suggesting DHRSX regulates the process rather than the initiation of autophagy. To investigate this relationship, researchers should:

• Monitor phosphorylation status of AKT and mTOR through Western blotting

• Assess Beclin1 protein levels in cells with manipulated DHRSX expression

• Use AKT/mTOR pathway inhibitors to determine if they mimic or enhance DHRSX effects

What are the optimal conditions for detecting DHRSX with antibodies in Western blot applications?

For successful Western blot detection of DHRSX:

• Use validated cell lines (HeLa, HepG2, A549) as positive controls

• Begin with the recommended antibody dilution (1:500-1:2000) and optimize as necessary

• Be aware that the observed molecular weight may differ from the calculated 36 kDa due to post-translational modifications or the presence of the intact signal peptide

• Include appropriate loading controls and consider both reducing and non-reducing conditions

• For detection of secreted DHRSX, concentrate cell culture supernatants before analysis

How can researchers optimize DHRSX antibody performance in immunohistochemistry applications?

For optimal IHC results with DHRSX antibodies:

• Start with validated tissue samples (human liver cancer, human lung cancer) when establishing protocols

• Perform antigen retrieval optimization to maximize specific epitope detection

• Use the recommended antibody dilution range (1:25-1:100) as a starting point

• Extend blocking steps to minimize non-specific binding

• Include positive and negative controls in each experiment

• Optimize incubation times and temperatures for both primary and secondary antibodies

What approach should be used to validate DHRSX knockdown experiments?

When designing DHRSX knockdown experiments:

• Use validated siRNA sequences targeting DHRSX:

  • DHRSX-homo-523: 5' GACCAACCUUCUCUUGGAUTT 3' (sense) and 5' AUCCAAGAGAAGGUUGGUCTT 3' (antisense)

  • DHRSX-homo-988: 5' GCAGCUGUGGUCUAAGAGUTT 3' (sense) and 5' ACUCUUAGACCACAGCUGCTT 3' (antisense)

• Transfect cells using Lipofectamine™ 2000 following manufacturer's protocols

• Confirm knockdown efficiency at both mRNA level (qRT-PCR) and protein level (Western blot)

• Include appropriate negative control siRNAs

• Assess functional consequences by measuring autophagy markers (LC3-II, p62) under both basal and starvation conditions

How can DHRSX recombinant proteins be utilized in functional studies?

Recombinant DHRSX proteins provide powerful tools for investigating function:

• GST-DHRSX fusion protein can be used for treatment experiments to assess direct effects on autophagy markers

• When applying recombinant protein to cells (e.g., HeLa, U2OS):

  • Begin with concentrations established in published literature

  • Include appropriate controls (e.g., GST protein alone)

  • Monitor multiple readouts (LC3-II levels, GFP-LC3 punctuation, electron microscopy of autophagic structures)

• For protein interaction studies, consider using tagged recombinant DHRSX (e.g., DHRSX-myc-his) for affinity purification

• Purified recombinant DHRSX can be used in enzymatic assays to investigate its oxidoreductase activity

What experimental approaches can elucidate DHRSX's potential role in disease processes?

Given DHRSX's involvement in autophagy, several approaches can explore its role in disease:

• Expression analysis across disease tissues, particularly those with known autophagy dysregulation (cancer, neurodegenerative disorders)

• Correlation studies between DHRSX expression/activity and disease progression markers

• In vitro disease models with DHRSX overexpression or knockdown to assess impact on disease-relevant phenotypes

• Investigation of DHRSX genetic variants in patient populations

• Modulation of DHRSX as a potential therapeutic approach in conditions with impaired autophagy

How might researchers investigate the tissue-specific functions of DHRSX?

To explore tissue-specific DHRSX functions:

• Compare expression levels across tissues using qRT-PCR, Western blot, and IHC

• Analyze tissue-specific effects of DHRSX modulation on autophagy and other cellular processes

• Examine potential tissue-specific interaction partners through co-immunoprecipitation and mass spectrometry

• Investigate regulatory mechanisms controlling tissue-specific expression patterns

• Consider the impact of the gene's unique chromosomal location (pseudoautosomal region) on expression in different tissues and between sexes

What are the optimal storage conditions for maintaining DHRSX antibody efficacy?

For maximum antibody stability and performance:

• Store antibodies at -20°C for long-term storage (valid for approximately 12 months)

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• Most commercial DHRSX antibodies are supplied in phosphate buffered solution (pH 7.4) containing 50% glycerol and 0.05% stabilizer

• Upon receipt of shipped antibodies (typically with ice packs), immediately store at the recommended temperature

• For short-term use, antibodies can typically be stored at 4°C for up to one week

What controls should be included when testing a new batch of DHRSX antibody?

When validating new antibody batches:

• Include positive control samples with known DHRSX expression (HeLa, HepG2, A549 cells)

• Run negative controls (samples with minimal or no DHRSX expression)

• Perform peptide competition assays to confirm specificity

• Compare results with previous antibody lots using standardized samples

• Test multiple applications (WB, IHC, IP) if the antibody is intended for multiple uses

• Consider testing recombinant DHRSX as a definitive positive control

How can researchers distinguish between experimental artifacts and true DHRSX detection?

To differentiate authentic DHRSX signals from artifacts:

• Be aware that the observed molecular weight may not match the calculated 36 kDa precisely

• Multiple bands may appear if different modified forms are present simultaneously

• Protein mobility can be affected by:

  • Post-translational modifications

  • Alternative splicing

  • Protein-protein interactions

  • Sample preparation conditions

• Use multiple antibodies targeting different DHRSX epitopes when possible

• Include appropriate controls and standardized samples in each experiment

• Consider the biological context and expected expression patterns when interpreting results