CPEB2 Antibody

What is CPEB2 and what are its key biological functions?

CPEB2 is an RNA-binding protein belonging to the CPEB family that regulates mRNA translation and stability. It contains RNA recognition motifs (RRMs) that bind to cytoplasmic polyadenylation elements (CPEs) in the 3'-UTR of target mRNAs.

CPEB2 functions include:

• Translational regulation of stored mRNAs in transcriptionally inactive haploid spermatids

• Tumor suppression in glioma by upregulating p21 mRNA translation and inhibiting cell proliferation

• Thermogenesis activation in brown adipose tissue by regulating Ucp1 mRNA translation

• Hormone sensing in mammary epithelial cells by controlling protein expression from ER/PR-regulated transcripts

• Lung development through PDGFRα mRNA translation in myofibroblast progenitors

What are the known isoforms of CPEB2 and their functional differences?

CPEB2 exists in multiple splice variants, with CPEB2A and CPEB2B being the most well-characterized:

The inclusion of exon 4 (encoding 30 amino acids) in CPEB2B drastically alters its function from a translational repressor to an activator . Up to 8 different isoforms have been reported for this protein, though many remain functionally uncharacterized .

What considerations are important when selecting CPEB2 antibodies?

When selecting CPEB2 antibodies, researchers should consider:

• Target epitope location: Antibodies targeting different regions of CPEB2 (N-terminal, middle region, or C-terminal) may have different specificities for detecting particular isoforms

• Species reactivity: Most commercial antibodies react with human, mouse, and rat CPEB2, but cross-reactivity varies among products

• Isoform specificity: Whether the antibody can distinguish between CPEB2A and CPEB2B if studying isoform-specific functions

• Application compatibility: Whether the antibody has been validated for your specific application (WB, ELISA, IF)

• Polyclonal vs. monoclonal: Polyclonal antibodies provide broader epitope recognition while monoclonals offer higher specificity

What validation strategies ensure CPEB2 antibody specificity?

To validate CPEB2 antibody specificity:

• Knockout/knockdown controls: Compare immunodetection between wild-type and CPEB2-knockout/knockdown samples. Only specific signals will disappear in knockout samples

• Recombinant protein controls: Test antibody against purified recombinant CPEB2 protein of known concentration

• Epitope blocking: Pre-incubate antibody with the immunizing peptide to confirm signal suppression

• Multiple antibody comparison: Use antibodies recognizing different CPEB2 epitopes to confirm consistent detection patterns

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of detected proteins by mass spectrometry after IP with the CPEB2 antibody

What are the optimal protocols for CPEB2 detection by Western blot?

Recommended Western Blot Protocol for CPEB2:

• Sample preparation:

  • Extract proteins using RIPA buffer with protease inhibitors

  • Human CPEB2 has a molecular weight of approximately 64.9 kDa

  • Include phosphatase inhibitors if studying phosphorylation status

• Gel electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membranes (recommended over nitrocellulose for CPEB2)

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary CPEB2 antibody at 1:500-1:5000 dilution (varies by supplier) overnight at 4°C

  • Wash 3x with TBST

  • Incubate with appropriate secondary antibody at 1:5000-1:10000 for 1 hour at room temperature

• Detection considerations:

  • CPEB2 expression may be tissue-dependent; BAT and neuronal tissues typically show higher expression

  • When detecting isoforms, longer running gels may be needed to distinguish the small size difference between CPEB2A and CPEB2B

How can researchers effectively use CPEB2 antibodies for immunofluorescence?

Immunofluorescence Protocol for CPEB2:

• Sample preparation:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • For tissue sections, optimize antigen retrieval methods as CPEB2 detection can be challenging in certain tissues

• Antibody incubation:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS for 10 minutes

  • Block with 5% normal serum (matching secondary antibody host) in PBS for 1 hour

  • Incubate with CPEB2 antibody at 1:50-1:200 dilution overnight at 4°C

  • Wash 3x with PBS

  • Incubate with fluorochrome-conjugated secondary antibody at 1:200-1:500 for 1 hour

• Visualization notes:

  • CPEB2 typically shows cytoplasmic localization

  • Co-staining with markers of RNA granules may be informative as CPEB2 can localize to these structures

  • Using Alexa Fluor 488-conjugated secondary antibodies has shown good results according to validation data

What approaches are effective for studying CPEB2's interaction with target mRNAs?

To study CPEB2-mRNA interactions:

• RNA immunoprecipitation (RIP):

  • Has successfully identified 169 CPEB2-bound mRNAs in mammary epithelial cells

  • CPEB2 targets show enrichment in canonical CPEs (UUUUA1-2U) in their 3'UTRs

  • Validated targets include Creb1, Ccnd1, and Rankl mRNAs in mammary epithelial cells

  • Essential to include appropriate CPEB2 knockout controls to confirm specificity

• Reporter assays for translational regulation:

  • Construct reporter plasmids containing firefly luciferase fused to the 3'UTR of potential CPEB2 targets

  • Compare reporter expression with or without CPEB2 overexpression/knockdown

  • This approach confirmed CPEB2-mediated regulation of Ucp1 and human UCP1 translation

• Polysome profiling:

  • To determine if CPEB2 affects translation efficiency of target mRNAs

  • Compare distribution of target mRNAs in polysome fractions between CPEB2 wild-type and knockout/knockdown samples

How can researchers investigate differential functions of CPEB2 isoforms?

To study isoform-specific functions:

• Isoform-specific knockdown:

  • Design siRNAs targeting the exon 4 junction in CPEB2B or the exon 3-5 junction in CPEB2A

  • RNA-seq analysis of cells with isoform-specific knockdown revealed that:

    • CPEB2B downregulation inhibited EMT and hypoxic response pathways

    • CPEB2A downregulation did not affect these pathways

• Isoform-specific overexpression:

  • Express FLAG-tagged CPEB2A or CPEB2B constructs

  • Shown to have opposite effects on HIF1α and TWIST1 protein expression without affecting mRNA levels

• Translation efficiency measurement:

  • Use nascent protein synthesis assays (e.g., SUnSET method) to compare how CPEB2A versus CPEB2B affects translation of target mRNAs

  • CPEB2A decreases while CPEB2B enhances nascent protein synthesis of targets

• Functional readouts:

  • Anoikis resistance assays showed CPEB2B promotes survival while CPEB2A suppresses it

  • Metastasis assays in orthotopic mouse models demonstrated CPEB2B enhances metastatic potential

What methodologies can assess CPEB2's role in tissue-specific functions?

For studying tissue-specific CPEB2 functions:

• Conditional knockout models:

  • Tissue-specific Cre-driven CPEB2 knockout mice have revealed:

    • BAT-specific role in thermogenesis regulation through Ucp1 translation

    • Lung-specific role in alveologenesis through PDGFRα expression

    • Mammary gland-specific role in hormone sensing

• ex vivo tissue explant cultures:

  • Mammary gland explants from CPEB2 KO mice showed defective ductal elongation in response to estrogen/progesterone

• Cell type isolation and analysis:

  • Flow cytometry sorting (e.g., using Sca1/CD49b markers for mammary epithelial subpopulations)

  • Single-cell RNA-seq data analysis to identify cell types expressing CPEB2 (e.g., LungGENS database showed CPEB2 expression in AECIs, AECIIs, and MYFs in lung tissue)

• Physiological functional assays:

  • CPEB2-KO mice showed pulmonary dysfunction with decreased elastance and resistance, resembling emphysema-like conditions

  • CL316243-induced thermogenesis measurements revealed CPEB2's role in brown fat activation

How should contradictory results with different CPEB2 antibodies be interpreted?

When facing contradictory results:

• Epitope accessibility issues:

  • Different fixation methods may affect epitope accessibility

  • Some studies reported difficulties obtaining specific CPEB2 immunostaining in certain tissues despite detection by Western blot

• Isoform recognition differences:

  • Antibodies raised against different regions may preferentially detect certain isoforms

  • N-terminal antibodies may detect both CPEB2A and CPEB2B, while those targeting exon 4 would be specific for CPEB2B

• Resolution steps:

  • Validate with genetic approaches (knockdown/knockout)

  • Use multiple antibodies targeting different epitopes

  • Compare with mRNA expression data

  • Consider tissue-specific post-translational modifications that might mask epitopes

What are common technical challenges when working with CPEB2 antibodies?

Common challenges include:

• Tissue fixation sensitivity:

  • CPEB2 detection in tissue sections often requires optimization of antigen retrieval methods

  • Some researchers failed to obtain specific CPEB2 immunostaining in pulmonary sections despite trying different protocols

• Distinguishing isoforms:

  • The small size difference between CPEB2A and CPEB2B (30 amino acids) requires optimized gel resolution

  • Consider using Phos-tag gels if phosphorylation status affects mobility

• Abundance variability:

  • CPEB2 expression varies significantly across tissues and cell types

  • Expression may be induced under specific conditions (e.g., stress, differentiation)

  • In human samples, CPEB2 mRNA levels were similar between mouse and human tissues, but human UCP1 mRNA was only 1/30 to 1/300 of mouse levels

• Solutions:

  • Include appropriate positive control tissues (brain, BAT)

  • Consider concentration of samples when expression is low

  • Use sensitive detection methods (ECL-Plus or fluorescent secondary antibodies)

What are emerging areas of CPEB2 antibody applications in disease research?

Recent research applications include:

• Cancer biology:

  • CPEB2 is significantly downregulated in glioma patient cohorts

  • CPEB2A/B ratio is altered in aggressive forms of breast cancer

  • CPEB2 antibodies can help identify tumors likely to develop metastatic potential based on isoform expression

• Developmental disorders:

  • CPEB2-KO mice surviving to adulthood show pulmonary dysfunction resembling emphysema-like conditions

  • Analysis of GEO datasets found decreased CPEB2 mRNA in umbilical cords of BPD (bronchopulmonary dysplasia) infants

• Metabolic diseases:

  • CPEB2's role in thermogenesis suggests potential applications in obesity research

  • SNPs in human UCP1 CPE sites (rs538981371, rs940802197) disrupt CPEB2-activated translation

How can CPEB2 antibodies contribute to understanding translational regulation mechanisms?

CPEB2 antibodies enable:

• Mapping of regulatory complexes:

  • Identification of proteins interacting with different CPEB2 isoforms

  • Understanding how exon 4 inclusion alters CPEB2's interaction with translational machinery

• Target identification:

  • RIP-seq experiments identified 169 CPEB2-bound mRNAs in mammary epithelial cells

  • Targets are enriched in breast cancer-related genes and PI3K-Akt signaling pathway components

• Mechanistic insights:

  • CPEB2A acts as a translational repressor while CPEB2B functions as a translational activator

  • This dual function may involve differential recruitment of polyadenylation factors