CAMK2N1 Antibody

What is CAMK2N1 and why is it significant in research?

CAMK2N1 functions as a tumor suppressor gene in several cancers, particularly prostate cancer and glioma. It inhibits cell proliferation, migration, and invasion while promoting apoptosis. The significance of CAMK2N1 lies in its regulatory role in critical signaling pathways, including PI3K/AKT and MEK/ERK pathways, and its involvement in androgen receptor (AR) signaling . Research has shown that reduced expression of CAMK2N1 is associated with cancer progression, making it an important target for cancer studies and potential therapeutic development.

What are the common applications for CAMK2N1 antibodies in research?

CAMK2N1 antibodies are employed in several laboratory techniques:

• Western blot (WB) analysis for protein expression quantification

• Immunohistochemistry (IHC) for tissue localization

• Immunofluorescence (IF) for cellular localization

• Chromatin immunoprecipitation (ChIP) for studying protein-DNA interactions

• Enzyme-linked immunosorbent assay (ELISA) for protein quantification

These applications allow researchers to investigate CAMK2N1 expression patterns, localization, and interactions with other molecules in various experimental contexts .

What are the most reliable sample types for CAMK2N1 antibody experiments?

Based on the search results, CAMK2N1 antibodies have been successfully used with:

• Human samples: Particularly prostate cancer cell lines (LNCaP, DU145, C4-2) and tissues

• Mouse samples: Brain tissue is particularly suitable for immunohistochemistry

• Cell lysates: Whole cell lysates from various cancer cell lines

For IHC applications, proper antigen retrieval is crucial, with TE buffer (pH 9.0) or citrate buffer (pH 6.0) recommended for optimal results .

What are the recommended protocols for CAMK2N1 antibody in Western blot applications?

For Western blot using CAMK2N1 antibodies:

• Cell preparation: Pellet cells and lyse in buffer supplemented with protease inhibitor cocktail

• Protein separation: Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

• Transfer: Transfer proteins to polyvinylidene fluoride (PVDF) membranes

• Blocking: Incubate membranes with 5% non-fat milk in TRIS-buffered saline with 2.5% Tween-20 for 1 hour at room temperature

• Primary antibody: Incubate with anti-CAMK2N1 antibody at 4°C overnight

• Secondary antibody: Incubate with appropriate secondary antibody

• Detection: Use standard chemiluminescence detection methods

The expected molecular weight for CAMK2N1 is approximately 9-10 kDa .

How should immunohistochemistry experiments with CAMK2N1 antibodies be conducted?

For optimal IHC results with CAMK2N1 antibodies:

• Sample preparation: Fix tissues and embed in paraffin, then prepare 4μm sections

• Antigen retrieval: Use TE buffer pH 9.0 (preferred) or alternatively citrate buffer pH 6.0

• Blocking: Block with appropriate serum (e.g., goat serum)

• Primary antibody: Use anti-CAMK2N1 antibody at dilutions of 1:200-1:800

• Secondary antibody: Apply appropriate secondary antibody

• Visualization: Use standard chromogenic or fluorescent detection methods

• Counterstaining: Counterstain nuclei with DAPI for fluorescence or hematoxylin for brightfield

Mouse brain tissue has shown reliable positive staining and can serve as a positive control .

What are the key considerations for chromatin immunoprecipitation (ChIP) assays involving CAMK2N1?

For ChIP assays investigating CAMK2N1 interactions:

• Cross-linking: Cross-link cells with 1% formaldehyde at 37°C for 10 minutes

• Cell lysis: Lyse cells for 1 hour on ice

• DNA shearing: Sonicate to shear DNA into appropriate fragment sizes

• Immunoprecipitation: Use ChIP-grade antibodies against CAMK2N1 or interacting proteins

• Controls: Include IgG antibody as a negative control

• DNA purification: After cross-link reversal with 5M NaCl (65°C for 4 hours), purify DNA

• Analysis: Perform PCR analysis on the enriched DNA fragments

Calculate fold enrichment by setting the value of the IgG control sample to 1 .

How does CAMK2N1 expression correlate with cancer progression and prognosis?

Research indicates that CAMK2N1 functions as a tumor suppressor in multiple cancer types:

• Prostate Cancer:

  • Downregulation of CAMK2N1 correlates with prostate cancer progression

  • CAMK2N1 inhibits prostate cancer cell proliferation, migration, and invasion

  • Loss of CAMK2N1 contributes to castration resistance in prostate cancer

  • Re-introduction of CAMK2N1 can sensitize castration-resistant cells to anti-androgen therapy

• Glioma:

  • Overexpression of CAMK2N1 indicates good prognosis in glioma patients

  • CAMK2N1 regulates glioma cell proliferation and apoptosis

In experimental models, knockdown of CAMK2N1 increased tumor volume and weight in xenograft models, while tumors with CAMK2N1 knockdown showed reduced expression of pro-apoptotic factors (p21, Bax) and increased proliferation markers (Ki67) .

What is the relationship between CAMK2N1 and DNA methylation in cancer?

CAMK2N1 and DNA methylation interact in a significant manner in cancer:

• Promoter hypermethylation contributes to CAMK2N1 downregulation:

  • In prostate cancer, CAMK2N1 is highly methylated compared to normal prostate epithelial cells

  • Bisulfite sequencing shows increased methylation at CpG sites in the CAMK2N1 promoter

  • Treatment with demethylating agent 5-Aza-CdR can restore CAMK2N1 expression

• Regulatory feedback loop with DNMT1:

  • DNMT1 (DNA methyltransferase 1) downregulates CAMK2N1 through promoter methylation

  • CAMK2N1 inhibits DNMT1 expression via the AKT or MEK/ERK signaling pathways

  • This creates a regulatory feedback mechanism affecting cancer progression

This relationship suggests targeting DNA methylation could be a therapeutic approach to restore CAMK2N1 expression in cancers where it is silenced.

How does CAMK2N1 interact with androgen receptor signaling in prostate cancer?

CAMK2N1 and androgen receptor (AR) form an auto-regulatory negative feedback loop:

• CAMK2N1 inhibits AR expression and activity:

  • Overexpression of CAMK2N1 reduces AR protein and mRNA levels

  • CAMK2N1 inhibits AR transactivation of target genes (PSA, TMPRSS2)

  • ChIP assays show CAMK2N1 reduces AR binding to enhancer elements of target genes

• AR regulation of CAMK2N1:

  • Androgen stimulation with R1881 affects CAMK2N1 expression

  • AR signaling influences CAMK2N1's effects on cell proliferation and cycle progression

• Functional consequences:

  • Knockdown of CAMK2N1 increases AR-dependent cell growth

  • CAMK2N1 depletion reduces sensitivity to AR antagonist Casodex

  • Re-expression of CAMK2N1 can restore sensitivity to anti-androgen therapy

This interaction has implications for developing treatments for castration-resistant prostate cancer.

What are the mechanisms by which CAMK2N1 regulates the PI3K/AKT and MEK/ERK pathways?

CAMK2N1 acts as a regulator of these critical signaling pathways:

• PI3K/AKT pathway:

  • Downregulation of CAMK2N1 increases phosphorylation and activation of AKT

  • CAMK2N1 overexpression reduces AKT phosphorylation

  • AKT inhibitors can abrogate the effects of CAMK2N1 knockdown, confirming pathway specificity

• MEK/ERK pathway:

  • CAMK2N1 suppresses the MEK/ERK signaling pathway

  • Knockdown of CAMK2N1 induces phosphorylation-mediated activation of MEK/ERK

  • ERK inhibitor U0126 can reverse the effects of CAMK2N1 knockdown

• Downstream effects:

  • These pathways mediate CAMK2N1's regulation of cell proliferation, apoptosis, and DNMT1 expression

  • Experimental evidence shows inhibitors of these pathways can reverse phenotypic changes caused by CAMK2N1 knockdown

Understanding these mechanisms provides insight into how CAMK2N1 exerts its tumor-suppressive functions.

How can researchers effectively study the functional interactions between CAMK2N1 and DNMT1?

To investigate CAMK2N1-DNMT1 interactions, researchers should consider these approaches:

• Gene expression manipulation:

  • Use plasmid-based overexpression systems (e.g., pLenti-EF1a-FH-CMV-GFP vector for CAMK2N1)

  • Apply siRNA or shRNA for knockdown (e.g., pGV493-shCAMK2N1, target sequence: 5′-GCAAGCGGGTTGTTATTGA-3′)

  • Create stable cell lines through antibiotic selection (puromycin, blasticidin)

• Pathway inhibition studies:

  • Use AKT inhibitors (AKTi) or ERK inhibitors (U0126) to block specific signaling pathways

  • Measure DNMT1 expression changes at mRNA and protein levels following CAMK2N1 manipulation and pathway inhibition

• DNA methylation analysis:

  • Apply bisulfite sequencing to assess methylation status of CAMK2N1 promoter

  • Use methylation-specific PCR (MSP) to detect changes in methylation patterns

  • Employ pyrosequencing for quantitative methylation analysis

• ChIP assays:

  • Perform ChIP with anti-DNMT1 antibodies to assess DNMT1 binding to the CAMK2N1 promoter

  • Calculate fold enrichment compared to IgG controls

This multi-faceted approach enables comprehensive characterization of this important regulatory interaction.

What experimental models are most appropriate for studying CAMK2N1's role in tumor suppression?

Based on the research data, these experimental models provide valuable insights:

• In vitro cellular models:

  • Prostate cancer cell lines: LNCaP (androgen-dependent), DU145 and C4-2 (castration-resistant)

  • Glioma cell lines: U87

  • Gene manipulation: Stable knockdown or overexpression of CAMK2N1

  • Functional assays: Proliferation (MTT), migration (wound healing), invasion (Transwell), apoptosis, cell cycle analysis (FACS)

• In vivo xenograft models:

  • Cell lines: DU145 or C4-2 with CAMK2N1 knockdown/overexpression

  • Animal host: BALB/c nude mice (4-5 weeks old)

  • Implantation: Subcutaneous injection of 1×10^6 cells

  • Measurements: Tumor volume (weekly), tumor weight (endpoint)

  • Analysis: Immunohistochemistry for CAMK2N1, AR, pAKT, PSA, Bax, Bcl-2, p21, Ki67

• Clinical samples:

  • Tumor tissue microarrays for comparing expression across cancer stages

  • Patient-derived samples for correlating CAMK2N1 expression with clinical outcomes

  • FFPE (formalin-fixed paraffin-embedded) samples for IHC analysis

These models allow for comprehensive investigation from molecular mechanisms to physiological relevance.

What are common challenges with CAMK2N1 antibody specificity and how can they be addressed?

Researchers may encounter these specificity issues:

• Potential challenges:

  • Cross-reactivity with related proteins

  • Background staining in immunohistochemistry

  • Inconsistent detection of the target protein

  • Varying results across different applications

• Validation approaches:

  • Positive controls: Use mouse brain tissue for IHC validation

  • Negative controls: Include samples with CAMK2N1 knockdown

  • Antibody specificity: Verify using western blot to confirm detection at the expected molecular weight (9-10 kDa)

  • Multiple antibodies: Use antibodies raised against different epitopes for confirmation

• Application-specific optimization:

  • For IHC: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for antigen retrieval

  • For Western blot: Optimize antibody dilution (starting at 1:500)

  • For immunofluorescence: Include proper blocking steps to reduce background

Maintaining appropriate positive and negative controls in each experiment is crucial for ensuring antibody specificity.

How can researchers effectively design experiments to study CAMK2N1's effects on cell proliferation and apoptosis?

For robust experimental design:

• Cell proliferation assessment:

  • MTT assay: Measure metabolically active cells following CAMK2N1 manipulation

  • Cell counting: Direct quantification of cell numbers over time

  • Colony formation assay: Evaluate long-term proliferative capacity

  • Include both gain-of-function (overexpression) and loss-of-function (knockdown) approaches

  • Assess in presence/absence of relevant stimuli (e.g., R1881 for AR-positive cells)

• Cell cycle analysis:

  • FACS analysis to determine cell distribution across cell cycle phases

  • Compare G0/G1, S, and G2/M phase distributions

  • Analyze effects of CAMK2N1 manipulation under different conditions (e.g., with/without androgen)

• Apoptosis evaluation:

  • Measure expression of apoptotic markers (Bax, Bcl-2)

  • TUNEL assay for detecting DNA fragmentation

  • Annexin V/PI staining for early/late apoptosis quantification

  • Western blot analysis of cleaved caspases

• Statistical analysis:

  • Perform Student's t-test for comparing two groups

  • Use one-way ANOVA followed by Tukey's multiple comparison test for multiple groups

  • Present results as mean ± standard deviation from at least three independent experiments

This comprehensive approach ensures reliable and reproducible results when investigating CAMK2N1's functional roles.

What controls should be included when studying CAMK2N1 methylation in cancer research?

When investigating CAMK2N1 methylation, include these essential controls:

• Cell line controls:

  • Positive methylation control: Cancer cell lines with known CAMK2N1 hypermethylation (e.g., PCa cells)

  • Negative methylation control: Normal prostate epithelial cells with low methylation

  • Treatment control: Cells treated with demethylating agent 5-Aza-CdR

• Technical controls for bisulfite conversion:

  • Unconverted DNA control: To verify conversion efficiency

  • Fully methylated DNA standard: Commercial methylated DNA as positive control

  • Unmethylated DNA standard: As negative control

  • No-template control: To detect contamination

• Methylation analysis controls:

  • For MSP: Both methylated and unmethylated primer sets

  • For bisulfite sequencing: Multiple clones (minimum 10) should be sequenced

  • For pyrosequencing: Include internal quality controls for bisulfite conversion

• Functional validation:

  • DNMT1 knockdown: To confirm methylation-dependent regulation

  • Gene expression correlation: Verify inverse relationship between methylation and expression

  • Genome-wide methylation assessment: To distinguish specific from global effects

These controls ensure the reliability and specificity of methylation findings in CAMK2N1 research.