CPNE3 Antibody

What is CPNE3 and why is it significant in cancer research?

CPNE3 is a member of the calcium-dependent phospholipid-binding protein family that has emerged as an important molecule in cancer biology. It is significantly upregulated in several cancer types, including GBM and NSCLC, and its overexpression is associated with poor prognosis. In GBM, CPNE3 promotes cell proliferation and inhibits apoptosis via the PI3K/AKT pathway . In NSCLC, CPNE3 enhances tumor cell migration and invasion through interactions with phosphorylated ErbB2 (pErbB2) and RACK1, activating the focal adhesion signaling pathway . The clinical significance makes CPNE3 a valuable target for cancer research and potential therapeutic interventions.

How does CPNE3 expression vary across different cancer types?

Expression analysis shows that CPNE3 is significantly upregulated in GBM tissues compared to adjacent normal tissues . Similarly, in NSCLC, CPNE3 is expressed at higher levels compared to corresponding non-cancerous lung tissues . Importantly, CPNE3 expression levels correlate with advanced TNM stages in NSCLC (p=0.024) and tumor size (p=0.048) . High levels of CPNE3 RNA are remarkably associated with poor outcomes (p<0.001) in lung cancer patients . When studying different cell lines, glioma cell lines (A172, T98G, U251, and U87) all exhibit significantly higher CPNE3 mRNA and protein expression compared to normal human glial cell line HEB .

What are the optimal antibody dilutions for detecting CPNE3 in different experimental applications?

Based on published research protocols, the following dilutions have shown effective results:

• For Western blotting: anti-CPNE3 antibody at 1:500 dilution (Abcam, Ab236606)

• For immunoprecipitation: Concentration depends on protein amount (typically using 3000 μg of total protein)

• For immunofluorescence: Dilutions must be optimized based on specific tissue type and fixation method

Always perform antibody titration experiments to determine optimal concentration for your specific experimental conditions and cell types.

What are the recommended protocols for CPNE3 knockdown experiments?

For effective CPNE3 knockdown studies, researchers have successfully used RNA interference approaches. Specifically:

• shRNA-mediated knockdown:

  • Validated target sequences:

    • shCPNE3-1 (788-806): GGTGGAGTGTTATGATTAT

    • shCPNE3-2 (1397-1415): GCAGACAGCTTCTCAATAT

    • shCPNE3-3 (1465-1483): CCAGACAAGCTATAGTTAA

  • These sequences should be cloned into appropriate lentiviral vectors (e.g., pLKO.1-puro)

  • Transfect packaging cells (HEK-293T) using Lipofectamine 2000

  • Collect viral supernatant after 48 hours and use to infect target cells

  • Validate knockdown efficiency by qPCR and western blot

• siRNA approach:

  • Design or purchase validated siRNAs targeting CPNE3

  • Transfect using Lipofectamine 2000 or similar reagent

  • Perform functional assays 48-72 hours post-transfection

Always include non-silencing controls (shNC or siNC) in all experiments for accurate comparison.

How should researchers design CPNE3 overexpression experiments?

For CPNE3 overexpression studies, the following protocol has proven effective:

• Obtain full-length CPNE3 cDNA and clone it into an appropriate lentiviral expression vector (e.g., pWPXL)

• Co-transfect the constructed plasmids and lentiviral packaging vectors into HEK-293T cells

• Collect viral supernatant 48 hours post-transfection

• Infect target cells (e.g., SPC-A-1, H1299, T98G) with the viral supernatant

• Validate overexpression by qPCR and western blot analysis

• Include empty vector controls (Vector) in all experiments

When selecting cell lines, consider using those with relatively low endogenous CPNE3 expression (e.g., T98G for glioma studies) to better observe the effects of overexpression .

What primer sequences are optimal for qPCR analysis of CPNE3 expression?

The following validated primer sequences have been used successfully for CPNE3 expression analysis:

CPNE3: Forward 5'-CATTGTAGAGGCGTATCG-3', Reverse 5'-CCATCACCATCCAGAAAC-3'

For normalization, GAPDH has been used effectively:
GAPDH: Forward 5'-AATCCCATCACCATCTTC-3', Reverse 5'-AGGCTGTTGTCATACTTC-3'

Always normalize CPNE3 expression to appropriate housekeeping genes and validate primer efficiency through standard curve analysis prior to experimental use.

How can researchers investigate CPNE3's role in the PI3K/AKT signaling pathway?

To study CPNE3's involvement in the PI3K/AKT pathway:

• Gene Set Enrichment Analysis (GSEA):

  • Analyze correlation between CPNE3 expression and PI3K-AKT-mTOR signaling pathway genes using cancer databases (e.g., TCGA)

• Protein phosphorylation analysis:

  • Assess phosphorylation status of PI3K and AKT in cells with altered CPNE3 expression

  • Use specific antibodies: anti-p-PI3K (1:1000, Abcam, Ab182651), anti-PI3K (1:1000, Abcam, Ab133595), anti-AKT (1:1000, Cell Signaling Technology, #9272), anti-p-AKT (1:2000, Cell Signaling Technology, #4060)

  • Compare ratios of phosphorylated to total protein levels

• Pathway inhibition studies:

  • Treat CPNE3-overexpressing cells with PI3K/AKT inhibitors (e.g., LY294002)

  • Assess whether inhibition reverses CPNE3-induced phenotypes

  • In published studies, LY294002 application alleviated the proliferation enhancement induced by CPNE3 overexpression

• Apoptosis and proliferation markers:

  • Examine XIAP (anti-apoptotic) and Bim (pro-apoptotic) expression

  • Monitor PCNA and Ki67 as proliferation markers

What methods are effective for studying CPNE3 protein interactions?

Co-immunoprecipitation (Co-IP) has been successfully used to identify CPNE3 protein interactions:

• Protocol overview:

  • Prepare cell lysates (3000 μg protein recommended)

  • Pre-clear with protein A/G magnetic beads (2 hours)

  • Incubate with anti-CPNE3 antibody overnight at 4°C

  • Add fresh beads and incubate overnight

  • Wash beads with PBS containing 1% Triton X-100 (five times)

  • Elute bound proteins by heating with 2× protein loading buffer at 100°C for 10 min

  • Analyze by SDS-PAGE followed by immunoblotting or mass spectrometry

• Validation approaches:

  • Perform reverse Co-IP using antibodies against suspected interaction partners

  • Use recombinant tagged proteins for pulldown assays

  • Include appropriate IgG controls

  • Confirm interactions through additional methods (proximity ligation assay, FRET)

Through this approach, researchers identified interactions between CPNE3 and phosphorylated ErbB2 (pErbB2) and RACK1 in NSCLC cells .

How should researchers design in vivo experiments to study CPNE3 function?

For effective in vivo studies of CPNE3 function, consider:

• Xenograft model establishment:

  • Generate stable CPNE3 knockdown or overexpressing cell lines

  • Implant cells subcutaneously in immunodeficient mice (e.g., nude mice)

  • Monitor tumor growth rate over time (e.g., measure tumor dimensions with calipers)

  • At experiment endpoint, collect tumors for size/weight measurements and further analysis

• Tissue analysis:

  • Perform immunohistochemistry or immunofluorescence for:

    • CPNE3 expression confirmation

    • Proliferation markers (PCNA, Ki67)

    • Pathway activation markers (p-AKT)

  • Conduct H&E staining to assess morphological changes

  • Consider TUNEL assays for apoptosis detection

• Experimental controls:

  • Include vector control or non-silencing shRNA control groups

  • Match for cell passage number and injection technique

  • Consider sample size based on power analysis

  • Blind observers during measurements and analysis

In published research, CPNE3 knockdown in U251 cells significantly reduced xenograft tumor growth and size compared to control groups .

What are common challenges in CPNE3 detection and how can they be addressed?

Researchers frequently encounter several issues when working with CPNE3 antibodies:

• Background signal:

  • Increase blocking time/concentration (5% nonfat dry milk has been effective)

  • Optimize antibody dilution (starting with manufacturer recommendations)

  • Include additional washing steps (5× PBST washes recommended)

  • Consider alternative blocking agents (BSA, normal serum)

• Inconsistent knockdown efficiency:

  • Test multiple siRNA/shRNA sequences (at least 3 different targets)

  • Optimize transfection conditions for each cell line

  • Validate knockdown at both mRNA (qPCR) and protein (western blot) levels

  • Consider using pooled siRNAs to increase efficiency

• Variable expression across cell lines:

  • Characterize baseline CPNE3 expression before selecting cell models

  • For knockdown, choose high-expressing lines (e.g., U251 for glioma studies)

  • For overexpression, select low-expressing lines (e.g., T98G)

How can conflicting results between different experimental approaches be reconciled?

When encountering contradictory results when studying CPNE3:

• Verify antibody specificity:

  • Confirm antibody recognizes the correct isoform

  • Use positive and negative control samples

  • Consider validation by mass spectrometry

  • Test multiple antibodies targeting different epitopes

• Cross-validate functional experiments:

  • Employ both gain-of-function and loss-of-function approaches

  • Use multiple cell lines representing different cancer subtypes

  • Confirm in vitro findings with in vivo models when possible

  • Validate key observations using different methodological approaches

• Control for pathway crosstalk:

  • When studying signaling (e.g., PI3K/AKT), consider pathway crosstalk

  • Use specific pathway inhibitors to confirm direct effects

  • Monitor multiple pathway components simultaneously

  • Consider time-course experiments to capture dynamic responses

What quantification methods should be used for analyzing CPNE3 expression and its downstream effects?

For robust quantification of CPNE3 and related molecules:

• Western blot quantification:

  • Use appropriate loading controls (GAPDH at 1:2000 dilution has been validated)

  • Employ densitometry software for band intensity measurement

  • Present data as ratio to loading control or as fold change relative to control samples

  • Perform at least three independent experiments for statistical analysis

• Cell proliferation assays:

  • CCK-8 assay has been effectively used to measure CPNE3-mediated proliferation effects

  • Conduct measurements at multiple timepoints (24h, 48h, 72h)

  • Present data as mean ± standard deviation

  • Include appropriate statistical tests (Student's t-test for two-group comparisons)

• Apoptosis quantification:

  • Flow cytometry with Annexin V/PI staining provides quantitative apoptosis assessment

  • Report percentage of apoptotic cells (early + late apoptosis)

  • Confirm with molecular markers (XIAP, Bim) by western blot

  • For statistical significance, p<0.05 is typically considered significant

What are promising approaches for targeting CPNE3 in cancer therapy research?

Based on current knowledge, several strategies for targeting CPNE3 show therapeutic potential:

• Small molecule inhibitors:

  • Target CPNE3's calcium-binding domains

  • Disrupt protein-protein interactions (e.g., CPNE3-RACK1 interaction)

  • Combine with established PI3K/AKT inhibitors (e.g., LY294002)

• Gene therapy approaches:

  • Utilize validated shRNA sequences for CPNE3 knockdown

  • Explore CRISPR/Cas9-mediated gene editing

  • Investigate delivery systems for targeted tumor therapy

• Biomarker development:

  • Evaluate CPNE3 as a prognostic biomarker in cancer patients

  • Develop diagnostic tools based on CPNE3 expression

  • Study CPNE3 in liquid biopsies (circulating tumor cells, exosomes)

How might researchers investigate CPNE3's role in therapy resistance mechanisms?

To study CPNE3 in therapy resistance:

• Develop resistant cell models:

  • Generate cell lines with acquired resistance to standard therapies

  • Compare CPNE3 expression and pathway activation pre/post-resistance

  • Assess whether CPNE3 knockdown resensitizes resistant cells

• Combination strategies:

  • Test CPNE3 targeting in combination with established therapies

  • Focus on PI3K/AKT pathway inhibitors, which have shown synergistic effects

  • Evaluate both sequential and concurrent treatment regimens

• Patient-derived models:

  • Analyze CPNE3 expression in patient samples before and after treatment failure

  • Establish patient-derived xenografts from treatment-resistant tumors

  • Correlate CPNE3 levels with treatment response in clinical cohorts