CERS4 Antibody

What is CERS4 and what is its primary function in human cells?

CERS4 (Ceramide Synthase 4) is a 394-amino acid endoplasmic reticulum multi-pass membrane protein that plays a critical role in sphingolipid metabolism. It functions as a ceramide synthase that catalyzes the formation of ceramide from sphinganine and acyl-CoA substrates, with high selectivity toward long and very-long chains (C18:0-C22:0) as acyl donors . In cellular contexts, CERS4 is particularly involved in the production of sphingolipids containing N-linked stearoyl (C18) or arachidoyl (C20) ceramides in a fumonisin B1-independent manner . The protein has a calculated molecular weight of approximately 46.4 kDa and is part of the LASS (longevity assurance homolog) family that is highly conserved from yeasts to mammals.

How do I select the appropriate anti-CERS4 antibody for my research application?

When selecting an anti-CERS4 antibody, consider these key parameters:

• Target species reactivity: Verify the antibody's reactivity with your species of interest. For instance, antibody M30561 is specifically reactive to human CERS4 .

• Application compatibility: Ensure the antibody has been validated for your intended application:

  • Western blotting (WB)

  • Flow cytometry

  • Immunohistochemistry (IHC)

  • ELISA

  For example, the Boster Bio Anti-CERS4 Antibody (N-Term) has been validated for WB and Flow Cytometry applications .

• Epitope location: Consider whether you need an antibody targeting a specific region (e.g., N-terminal) based on your experimental goals. Some antibodies, like M30561, are specifically generated against the N-terminal region (amino acids 21-51) of human CERS4 .

• Clonality: Choose between polyclonal (broader epitope recognition) and monoclonal (higher specificity) based on your experimental needs. Many available CERS4 antibodies are polyclonal, such as the rabbit polyclonal antibody from OriGene (TA351343) .

• Validation data: Review provided validation images and positive control information. For example, the OriGene antibody recommends human fetal liver tissue, HT-29 and HUVEC cells, MCF-7 cells, and human hepatocellular carcinoma tissue as positive controls for Western blotting .

What are the optimal protocols for using anti-CERS4 antibodies in Western blotting?

For optimal Western blotting using anti-CERS4 antibodies, follow this methodology:

• Sample preparation:

  • Lyse cells in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100 or Nonidet P-40, plus protease and phosphatase inhibitors)

  • Incubate lysates on ice for 30 minutes

  • Centrifuge at 12,000 ×g for 15 minutes at 4°C to collect supernatants

  • Quantify protein using Bio-Rad Protein Assay Dye Reagent

• Gel electrophoresis and transfer:

  • Load 30-50 μg of heat-denatured protein

  • Separate on 10% SDS-PAGE

  • Transfer to nitrocellulose membrane

  • Block with 5% bovine serum albumin (BSA) in TBST for 1 hour

• Antibody incubation:

  • Dilute primary anti-CERS4 antibody at 1:500-1:2000 in blocking buffer

  • Incubate membrane overnight at 4°C on a shaking incubator

  • Wash with TBST

  • Incubate with appropriate secondary antibody

• Detection and analysis:

  • Develop using chemiluminescence

  • Expected band size: approximately 46 kDa

• Controls:

  • Positive controls: Human fetal liver tissue, HT-29 and HUVEC cells, MCF-7 cells, or human hepatocellular carcinoma tissue

How can I use CERS4 antibodies in studying skin and hair follicle development?

CERS4 antibodies are valuable tools for investigating skin and hair follicle development, particularly focusing on hair follicle stem cell (HFSC) lineage specification. Based on recent research methodologies :

• Tissue preparation and immunofluorescence:

  • Fix tissue biopsies with 4% PFA in PBS

  • Embed in paraffin and section

  • Deparaffinize using a graded alcohol series

  • Block in 10% normal goat serum

  • Incubate with anti-CERS4 antibody diluted in Dako Antibody Diluent overnight at 4°C

  • Detect with Alexa Fluor 488 or 568-conjugated secondary antibodies

  • Counterstain nuclei with DAPI

• Co-localization studies:

  • Combine CERS4 antibody with stem cell markers (CD34, Lhx2, Lgr5)

  • Use keratin markers (K14, K6, K15) to identify different epithelial components

  • Include Lef1 antibody to examine Wnt pathway activation

• Functional analysis through genetic models:

  • Generate or obtain CerS4 knockout or conditional knockout mice

  • Use epidermis-specific deletion (K14-Cre), SG-specific deletion (SCD3-Cre), or HFSC-specific deletion (Lgr5eGFP-CreERT2)

  • Compare changes in hair follicle development between control and CerS4-deleted tissues

• Organoid culture systems:

  • Isolate HFSCs using FACS (CD34+/integrin α6+)

  • Culture in appropriate 3D conditions

  • Monitor morphology and differentiation patterns

  • Use anti-CERS4 antibodies to track expression changes during differentiation

How can CERS4 antibodies be used to investigate the role of ceramide synthases in cancer progression?

CERS4 antibodies can be strategically employed to explore ceramide synthase roles in cancer through these methodologies:

• Expression analysis in cancer tissues:

  • Compare CERS4 expression between cancer and adjacent normal tissues using immunohistochemistry

  • Quantify expression differences using tissue microarrays

  • Correlate expression levels with clinicopathological parameters and survival data

• Functional studies using genetic manipulation:

  • Generate stable CERS4-overexpressing cell lines (e.g., MCF-7/CerS4 cells for breast cancer)

  • Create CERS4-knockdown models using lentivirus-mediated RNAi

  • Validate expression changes by Western blot using anti-CERS4 antibodies

  • Assess effects on proliferation, migration, invasion, and drug resistance

• Signaling pathway analysis:

  • Use Western blotting with anti-CERS4 and pathway-specific antibodies

  • In liver cancer studies, examine NF-κB pathway components

  • In breast cancer, assess Wnt/β-catenin pathway activation using anti-active-β-catenin antibodies alongside CERS4 detection

• In vivo tumor models:

  • Inject CERS4-modified cancer cells into immunodeficient mice

  • Monitor tumor growth parameters (volume, weight)

  • Harvest tumors for immunohistochemical analysis using anti-CERS4 antibodies

  • Correlate CERS4 expression with tumor aggressiveness

Example research finding: In liver cancer studies, silencing CERS4 with lentivirus-mediated RNAi significantly suppressed proliferation rates (P<0.001) and reduced tumor weight and volume in vivo, indicating CERS4 as an important regulator of liver cancer cell proliferation .

What methodologies can be used to study CERS4's role in inflammatory and immune responses?

To investigate CERS4's role in inflammatory and immune responses, implement these methodological approaches:

• T-cell specific studies:

  • Isolate primary T-cells from thymus or peripheral blood

  • Culture in appropriate medium (RPMI with supplements)

  • Stimulate with IL-2 (200 units/mL) and anti-CD2/3/28 activation beads

  • Analyze CERS4 expression changes during T-cell activation using antibodies

• Flow cytometry for immune cell profiling:

  • Prepare single-cell suspensions from tissues

  • Stain with CERS4 antibodies alongside immune cell markers

  • Gate immune cell subsets (CD45+, CD4+, GATA3+ for Th2; lineage−, Nkp46−, CD90.2+, GATA3+ for ILC2)

  • Quantify differences in immune cell populations between control and CERS4-deficient models

• Inflammatory disease models:

  • Generate tissue-specific CERS4 knockout models (e.g., T-cell-specific CerS4 depletion)

  • Induce inflammatory conditions

  • Monitor inflammation progression and resolution

  • Compare with human disease transcriptional profiles using GSEA

• Sphingolipid profiling in immune contexts:

  • Extract lipids from immune cells or tissues

  • Perform lipidomic analysis using LC-MS/MS

  • Correlate sphingolipid profiles with CERS4 expression levels

  • Identify specific ceramide species affected by CERS4 alteration

Research finding: T-cell-specific CerS4 depletion led to prolonged inflammation and prevention of resolution, resulting in higher tumor development. Flow cytometry analysis revealed a shift toward Th2 dominance in CerS4-deficient mice, associated with increased ILC2 cell numbers, mimicking features of human atopic dermatitis .

What are common issues with CERS4 antibodies and how can they be addressed?

Common issues with CERS4 antibodies and their solutions include:

• Non-specific binding:

  • Problem: Multiple bands appearing in Western blots

  • Solution: Increase blocking time/concentration (5% BSA in TBST); optimize antibody dilution (1:500-1:2000); include additional washing steps; use peptide competition assays with the immunizing peptide

• Weak signal:

  • Problem: Faint or no detection of CERS4

  • Solution: Ensure adequate protein loading (30-50 μg); optimize antibody concentration; increase incubation time (overnight at 4°C); use signal enhancement systems; verify sample preparation (inhibit proteases)

• Background issues in immunohistochemistry:

  • Problem: High background staining obscuring specific signals

  • Solution: Optimize blocking (10% normal goat serum); adjust antibody dilution (1:50-1:200); include additional washing steps; use antigen retrieval if needed

• Cross-reactivity with other CerS family members:

  • Problem: Antibody detecting multiple CerS proteins

  • Solution: Validate specificity using CERS4 knockout controls; select antibodies raised against unique regions; use peptide competition assays

• Storage and stability issues:

  • Problem: Decreased antibody performance over time

  • Solution: Store according to manufacturer recommendations (typically at -20°C); avoid freeze-thaw cycles by preparing small aliquots; add stabilizing proteins if needed

Quality control measures:

• Always include positive controls (e.g., human fetal liver tissue, HT-29 and HUVEC cells, MCF-7 cells)

• Include negative controls (secondary antibody only, isotype controls)

• Validate any new antibody lot against previous lots or using knockout/knockdown samples

How can I validate the specificity of anti-CERS4 antibodies?

To rigorously validate anti-CERS4 antibody specificity, employ these methodologies:

• Genetic knockout/knockdown controls:

  • Generate CERS4 knockout models or knockdown cell lines using lentivirus-mediated RNAi

  • Compare antibody reactivity between wild-type and CERS4-deficient samples

  • Absence of signal in knockout/knockdown samples confirms specificity

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Apply this mixture to parallel samples alongside untreated antibody

  • Specific signals should be blocked in the peptide-treated condition

• Orthogonal detection methods:

  • Correlate protein detection with mRNA levels using RT-qPCR

  • Confirm changes in CERS4 protein levels match transcriptional changes

  • Example methodology: Extract RNA using RNeasy Mini Kits, synthesize cDNA, perform real-time PCR using SYBR-Green Master Mix

• Multiple antibody validation:

  • Test different antibodies targeting distinct epitopes of CERS4

  • Compare detection patterns across antibodies

  • Consistent results across antibodies increase confidence in specificity

• Recombinant expression systems:

  • Overexpress tagged versions of CERS4 (e.g., HA-tagged CERS4)

  • Detect with both anti-CERS4 and anti-tag antibodies

  • Co-localization confirms specificity

• Mass spectrometry validation:

  • Immunoprecipitate using anti-CERS4 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm CERS4 as the predominant isolated protein

How do I interpret contradictory findings regarding CERS4 expression across different cancer types?

Interpreting contradictory CERS4 expression patterns across cancer types requires careful methodological consideration:

• Tissue-specific context analysis:

  • Different tissues have distinct baseline CERS4 expression levels

  • Compare cancer samples to matched normal tissues from the same organ

  • Consider cell-type specific expression within heterogeneous tissues

• Isoform and mutation analysis:

  • Examine whether antibodies detect all CERS4 isoforms

  • Consider potential RNA editing effects, as seen in pancreatic cancer where CERS4 RNA undergoes A-to-I editing resulting in exon 3 loss and a truncated protein

  • Determine if similar RNA editing occurs in other cancer types

• Methodological differences assessment:

  • Compare detection methods (IHC vs. Western blot vs. RNA-seq)

  • Analyze antibody epitope locations (N-terminal vs. C-terminal)

  • Consider sample preparation variations (fresh vs. FFPE tissues)

• Integrate multi-omics data:

  • Correlate protein expression with transcriptome data

  • Analyze sphingolipid profiles in relation to CERS4 expression

  • Examine genetic alterations (mutations, copy number variations) affecting CERS4

• Functional context interpretation:

  • In liver cancer: CERS4 shows oncogenic properties, with high expression promoting proliferation

  • In breast cancer: CerS4 overexpression exerts oncogenic effects via alterations in signaling, EMT, and chemoresistance

  • In colon cancer: CerS4 is downregulated, potentially through cell stress mechanisms

  These differences suggest tissue-specific roles and potential dual functions depending on cellular context.

How can CERS4 antibodies be used in identifying potential biomarkers for metabolic and inflammatory diseases?

CERS4 antibodies can be strategically employed for biomarker development in metabolic and inflammatory conditions through these approaches:

• Clinical sample analysis:

  • Analyze CERS4 expression in patient samples using immunohistochemistry or Western blotting

  • Compare expression patterns between:

    • Healthy controls and disease states

    • Different disease severities

    • Treatment responders and non-responders

• Correlation with genetic markers:

  • Genotype patients for CERS4 genetic variants (e.g., rs17160348)

  • Use antibodies to determine if protein expression correlates with genotype

  • Assess the relationship between CERS4 variants, protein expression, and disease manifestation

  Example from research: The T-allele of CERS4 rs17160348 was associated with higher risk of both obese and nonobese MASLD (OR: 1.95, 95% CI: 1.20–3.15; OR: 1.76, 95% CI: 1.08–2.86, respectively)

• Integration with metabolic profiles:

  • Combine CERS4 protein expression data with sphingolipid profiling

  • Correlate with metabolic parameters (lipid profiles, glucose metabolism)

  • Identify sphingolipid species that serve as functional biomarkers downstream of CERS4

  Research finding: Effects of CERS4 rs17160348 C allele on MASLD were influenced by levels of phosphatidylcholine, phosphatidic acid, sphingomyelin, and phosphatidylinositol

• Inflammatory biomarker panels:

  • Determine CERS4 expression in inflammatory conditions (e.g., atopic dermatitis)

  • Combine with established inflammatory markers

  • Assess correlation with disease severity and treatment response

  Finding: CerS4-deficient mice develop a Th2-dominated immune phenotype with transcriptional signatures sharing features with human atopic dermatitis

• Translational validation pipeline:

  • Begin with discovery in animal models using CERS4 antibodies

  • Validate in human samples across disease stages

  • Develop standardized assays with optimized antibody concentrations and protocols

  • Perform multi-center validation studies

This methodological approach enables the development of CERS4-related biomarkers with both diagnostic and prognostic value across metabolic and inflammatory disease spectrums.

What emerging technologies can enhance CERS4 antibody-based research?

Emerging technologies to enhance CERS4 antibody research include:

• Single-cell antibody-based technologies:

  • Implement mass cytometry (CyTOF) with CERS4 antibodies for high-dimensional single-cell analysis

  • Apply imaging mass cytometry to maintain spatial context while detecting CERS4 in tissue sections

  • Utilize single-cell Western blotting to analyze CERS4 expression heterogeneity within populations

• Proximity labeling techniques:

  • Employ BioID or APEX2 proximity labeling fused to CERS4

  • Use antibodies to detect CERS4 interactome components

  • Map protein-protein interactions within the sphingolipid metabolism network

• Advanced microscopy approaches:

  • Implement super-resolution microscopy with CERS4 antibodies to examine subcellular localization

  • Use live-cell imaging with nanobody-based detection systems

  • Combine with organelle markers to track CERS4 trafficking and localization

• Antibody engineering for expanded applications:

  • Develop bi-specific antibodies targeting CERS4 and other pathway components

  • Create antibody-drug conjugates for targeted therapy in CERS4-overexpressing cancers

  • Design intrabodies for live-cell tracking of CERS4 dynamics

• Organoid and 3D culture systems:

  • Apply CERS4 antibodies in complex organoid systems that better recapitulate in vivo conditions

  • Perform high-content imaging analysis to track CERS4 expression during differentiation

  • Correlate with functional outcomes in tissue-specific contexts

These technological advances will provide deeper insights into CERS4 biology and expand the utility of CERS4 antibodies beyond conventional applications.

How might targeting CERS4 lead to novel therapeutic approaches in cancer and inflammatory diseases?

The therapeutic potential of targeting CERS4 can be explored through these methodological approaches:

• Experimental therapeutic modalities:

  • Develop neutralizing antibodies against CERS4

  • Design small molecule inhibitors based on structure-activity relationships

  • Implement siRNA or antisense oligonucleotides for targeted CERS4 downregulation

  • Use CERS4 antibodies to evaluate target engagement and efficacy

• Disease-specific therapeutic approaches:

  • For cancer therapy:

    • In liver cancer: Investigate CERS4 inhibition to suppress proliferation and tumor growth

    • In breast cancer: Target CERS4 to overcome chemoresistance mechanisms

    • Use combination approaches with standard chemotherapies

  • For inflammatory conditions:

    • In atopic dermatitis: Modulate CERS4 to restore normal HFSC function and skin barrier

    • For T-cell mediated inflammation: Target CERS4 to regulate T-cell activation status

• Biomarker-guided therapeutic strategies:

  • Use CERS4 antibodies for patient stratification

  • Develop companion diagnostics to identify patients likely to benefit from CERS4-targeted therapies

  • Monitor treatment efficacy through CERS4 expression changes

• Preclinical validation methodologies:

  • Generate tissue-specific CERS4 knockout models

  • Perform pharmacological inhibition studies

  • Conduct patient-derived xenograft experiments

  • Assess effects on disease progression, inflammatory markers, and clinical endpoints

Research findings supporting therapeutic potential:

• In liver cancer, CERS4 silencing reduced tumor weight and volume in mouse models

• In MASLD, specific CERS4 genotypes (rs17160348) were associated with disease risk and severity

• In inflammatory skin conditions, CerS4 deletion led to atopic dermatitis-like phenotypes, indicating potential for modulation in treating inflammatory skin disorders