CYTL1 Antibody

What is CYTL1 and why is it important to use specific antibodies in research?

CYTL1 (Cytokine-like protein 1, also known as Protein C17 or C4orf4) is a 16 kDa secretory protein encoded by a gene located on human chromosome 4p15-p16 . It has 126 amino acid residues with characteristics of a secretory protein and is primarily expressed in CD34+ human mononuclear cells from bone marrow and cord blood, as well as chondrocytes .

When selecting CYTL1 antibodies for research, consider:

• Specificity: Most commercial CYTL1 antibodies show reactivity with human samples, while some cross-react with mouse and rat samples

• Applications: Different antibodies are validated for specific applications (WB, IHC, IF, ELISA)

• Clonality: Both polyclonal (e.g., PAC041166, 15856-1-AP) and monoclonal options are available

• Validation data: Review immunohistochemistry images and Western blot data showing the expected 16 kDa band

The importance of using specific antibodies lies in CYTL1's involvement in multiple physiological processes including chondrogenesis, immune regulation, and potential roles in cancer biology .

Immunohistochemistry (IHC):

• Recommended dilution: 1:20-1:200 for PACO41166 , 1:50-1:500 for 15856-1-AP

• Antigen retrieval: Use TE buffer pH 9.0; alternatively, citrate buffer pH 6.0

• Visualization: Example from published data shows strong staining in mouse lung and liver tissues

• Positive controls: Human testis tissue shows positive staining at 1:100 dilution

Western Blotting:

• Dilution: 1:500 is typically effective for most CYTL1 antibodies

• Expected molecular weight: 16 kDa primary band

• Sample preparation: Protein G purification enhances specificity

• Buffer system: 0.01M PBS, pH 7.4 with 50% Glycerol and 0.03% Proclin 300 as preservative

ELISA:

• Recommended dilution: 1:2000-1:10000

• Indirect ELISA protocol: Validated for measuring CYTL1 in cell culture supernatants

• Detection system: Anti-CYTL1 antibody (ab129767) at 1:40000 dilution followed by HRP-conjugated secondary antibody at 1:5000

How can researchers distinguish between intracellular and secreted forms of CYTL1?

Distinguishing between intracellular and secreted CYTL1 is crucial as they may have different functions:

• Structural differences:

  • Secreted CYTL1 contains a 1-22 amino acid signal peptide

  • Intracellular CYTL1 (ΔCYTL1) lacks this signal peptide

• Methodological approaches:

  • Cellular fractionation: Separate cytoplasmic and membrane fractions before Western blotting

  • Immunofluorescence: Use permeabilized vs. non-permeabilized conditions to distinguish localization

  • ELISA: Measure CYTL1 in cell culture supernatants (secreted) vs. cell lysates (intracellular)

• Functional differences:

  • Intracellular CYTL1 has been identified as a potential tumor suppressor that stabilizes NDUFV1 to prevent metabolic switching from OXPHOS to glycolysis in breast cancer

  • Secreted CYTL1 demonstrates chemotactic and pro-angiogenic effects

• Expression analysis:

  • Quantify CYTL1 in conditioned media to measure secretion rates

  • Compare with intracellular levels to determine secretion efficiency

In vitro models:

• Chondrocyte culture systems:

  • Primary chondrocytes or chondrogenic cell lines (ATDC5, SW1353)

  • Differentiation of mesenchymal stem cells into chondrocytes with CYTL1 supplementation or knockdown

  • 3D culture systems (pellet culture, alginate beads) to mimic cartilage environment

• Molecular analysis:

  • Monitor Sox9 and insulin-like growth factor 1 expression as downstream markers of CYTL1 activity

  • Analyze ECM components (collagen II, aggrecan) expression and deposition

  • Investigate TGF-beta-Smad and ERK signaling pathway activation

In vivo models:

• Cytl1 knockout mice:

  • Studies demonstrate that Cytl1 maintains cartilage homeostasis

  • Deletion of Cytl1 results in accelerated osteoarthritis progression

• Surgical models:

  • DMM (destabilization of the medial meniscus) model with Cytl1-/- mice

  • Analyze cartilage degeneration, osteophyte formation, and subchondral bone changes

Experimental readouts:

• Histological analysis:

  • Safranin O/Fast Green staining for proteoglycan content

  • Immunohistochemistry for CYTL1, Sox9, and ECM components

• Functional tests:

  • Compression testing of cartilage

  • Assessment of joint mobility and gait analysis in animal models

How can researchers investigate the interaction between CYTL1 and its receptor CCR2?

CCR2 (C-C chemokine receptor type 2) has been identified as a potential receptor for CYTL1 . To study this interaction:

Binding assays:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified CCR2 or CYTL1 on sensor chips

  • Measure binding kinetics and affinity constants (K₄, K₀ₙ, K₀ₙₙ)

• Co-immunoprecipitation:

  • Use anti-CYTL1 antibodies to pull down protein complexes

  • Detect CCR2 in the immunoprecipitates by Western blotting

  • Control experiments should include CCR2 blocking antibodies

Signaling studies:

• ERK pathway activation:

  • Treat cells with recombinant CYTL1 (0.1-10 ng/ml)

  • Measure phosphorylation of ERK by Western blotting or ELISA

  • Use CCR2 antagonists to confirm specificity of signaling

• Calcium flux assays:

  • Load cells with calcium-sensitive dyes

  • Monitor intracellular calcium changes upon CYTL1 stimulation

  • Compare responses in CCR2-expressing vs. CCR2-knockout cells

Functional validation:

• Cell migration assays:

  • Use Transwell chambers with recombinant CYTL1 as chemoattractant

  • Evaluate migration of CCR2-expressing vs. CCR2-deficient cells

  • Confirm specificity using neutralizing antibodies against CYTL1 or CCR2

• In vivo models:

  • Compare phenotypes of Cytl1-/- and CCR2-/- mice

  • Rescue experiments with wild-type or mutant proteins

What are the best strategies for validating CYTL1 antibody specificity?

Antibody validation is critical for generating reliable data. For CYTL1 antibodies:

Positive validation approaches:

• Recombinant protein controls:

  • Use CYTL1 recombinant protein (such as human CYTL1/C17 hFc Chimera)

  • Test multiple concentrations (5-10 μg/ml)

• Overexpression systems:

  • Transfect cells with CYTL1 expression vectors (e.g., pcDB-IgGκSP-CYTL1-6×His)

  • Compare signal in transfected vs. non-transfected cells

• Positive tissue controls:

  • Human testis tissue shows positive staining at 1:100 dilution

  • Mouse lung and liver tissues show strong immunoreactivity

Negative validation approaches:

• Knockdown/knockout controls:

  • siRNA for CYTL1 gene expression reduction

  • CRISPR/Cas9-mediated knockout cell lines

  • Cytl1-/- mouse tissues

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptides (e.g., P1 and P3 peptides)

  • Observe disappearance of specific signal

• Antibody dilution series:

  • Test serial dilutions to determine optimal working concentration

  • Evaluate signal-to-noise ratio

Cross-reactivity testing:

• Multiple species testing:

  • Verify cross-reactivity with mouse and rat CYTL1 if using in these models

  • Consider sequence homology when interpreting results

How do researchers interpret contradictory CYTL1 expression data between different experimental approaches?

When facing contradictory results:

Common causes of discrepancies:

• Antibody specificity issues:

  • Different epitope recognition between antibodies

  • Cross-reactivity with similar proteins

  • Solution: Use multiple antibodies targeting different epitopes

• Different isoforms:

  • Intracellular vs. secreted forms may show different patterns

  • Solution: Use isoform-specific detection methods

• Post-translational modifications:

  • Glycosylation or phosphorylation may affect antibody binding

  • Solution: Use different detection methods (e.g., mass spectrometry)

Reconciliation strategies:

• Multi-level analysis:

  Level	Method	Information Provided
  mRNA	qRT-PCR, RNA-seq	Transcriptional regulation
  Protein	Western blot, IHC, IF	Protein expression and localization
  Functional	Activity assays	Biological activity

• Context consideration:

  • CYTL1 expression varies by tissue type and disease state

  • Expression in STAD samples (n=375) differs from normal samples (n=32)

  • High vs. low expression correlates with patient survival in certain cancers

What role does CYTL1 play in cancer biology and how can researchers study this?

CYTL1 has both pro-oncogenic and tumor-suppressive roles depending on cancer type:

Oncogenic properties:

• CYTL1 expression in neuroblastoma correlates with cell proliferation, migration, and invasion

• In stomach adenocarcinoma (STAD), high CYTL1 expression correlates with poor prognosis (HR=1.981, p<0.001)

Experimental approaches:

• Expression modulation:

  • Overexpression of wild-type or mutant CYTL1 (with/without signal peptide)

  • siRNA knockdown or CRISPR knockout systems

  • Inducible expression systems to study temporal effects

• Functional assays:

  • Proliferation: CCK-8 assay for cell viability

  • Migration/invasion: Transwell or wound healing assays

  • Metabolism: Measure glucose uptake, lactate production, oxygen consumption

• Molecular mechanism studies:

  • Protein-protein interactions (e.g., CYTL1-NDUFV1, CYTL1-MDM2)

  • Signaling pathway analysis (ERK, TGF-beta-Smad)

  • Metabolic pathway analysis (OXPHOS vs. glycolysis)

• In vivo models:

  • Xenograft models with CYTL1-modulated cancer cells

  • Patient-derived xenografts

  • Transgenic mouse models with tissue-specific expression

Immunohistochemistry quantification:

• Semi-quantitative scoring:

  • Staining intensity (0-3+)

  • Percentage of positive cells (0-100%)

  • H-score calculation: ∑(intensity × percentage)

  • Examples from publications show varied staining patterns in different tissues

• Digital image analysis:

  • Whole slide scanning

  • Computer-assisted quantification of staining intensity

  • Segmentation of cellular compartments (membrane, cytoplasm, nucleus)

Western blot quantification:

• Densitometric analysis:

  • Normalize CYTL1 band intensity to loading controls (β-actin, GAPDH)

  • Use standard curves with recombinant CYTL1 protein

  • Example: Detecting ~19 kDa band in purified samples

ELISA-based quantification:

• Indirect ELISA protocol:

  • Validated for cell culture supernatants

  • Use standard curves with recombinant CYTL1

  • Detection range typically 0.1-10 ng/ml

What are the emerging research directions for CYTL1 beyond its role in chondrogenesis?

Recent research has expanded CYTL1's role beyond cartilage biology:

Reproductive biology:

• CYTL1 enhances endometrial cell adhesion to trophoblastic cells

• Application of 10 ng/ml CYTL1 significantly increases JAR spheroid adhesion (76.97±3.08% vs. 21.04±4.41% in controls)

• CYTL1 regulates LIF and HB-EGF expression in endometrial cells

Cardiovascular research:

• CYTL1 shows pro-angiogenic function in endothelial progenitors

• Potential therapeutic application in ischemic pathologies

• CYTL1's role in cardiac fibrosis and heart failure

Cancer metabolism:

• Intracellular CYTL1 stabilizes NDUFV1 to prevent metabolic switching in breast cancer

• CYTL1 competitively binds the N-terminal sequence of NDUFV1 to block MDM2-mediated degradation

• This increases NAD+ levels and attenuates LDHA phosphorylation

Immunomodulation:

• CYTL1 bears sequence homology to IL-2 and may contribute to immune homeostasis

• Research opportunities in inflammatory and autoimmune conditions

• Potential therapeutic target in diseases with immune dysfunction