At1g47840 Antibody

Target Protein: E2FB Transcription Factor

The At1g47840 gene encodes E2FB, a member of the E2F transcription factor family involved in regulating cell cycle progression and endoreduplication. Key characteristics include:

• Function: Promotes G1-to-S phase transition and suppresses endoreduplication under hormone-free conditions .

• Structure: Contains a DNA-binding domain and a dimerization domain for interaction with DP proteins .

Antibody Development and Validation

The antibody was generated using a recombinant protein fragment corresponding to the C-terminal region of E2FB (residues 249–end) .
Key steps in production:

Immunoblot Results:

• Detected endogenous E2FB in synchronized Arabidopsis cells.

• Showed correlation with CDKB1;1 protein levels during cell cycle progression .

Cell Cycle Regulation Studies

• Overexpression Effects: Elevated E2FB levels in transgenic BY-2 cell lines led to:

  • Sustained S-phase entry under stationary conditions.

  • Inhibition of endoreduplication (reduced 4C/8C DNA content) .

• Interaction with CDKs: E2FB overexpression correlated with constitutive high levels of CDKB1;1 protein, suggesting direct transcriptional regulation .

Experimental Workflow

1. Cell Synchronization: Aphidicolin treatment to arrest cells at G1/S phase.

2. Protein Analysis:

   • E2FB levels peaked during S and G2/M phases.

   • CDKB1;1 accumulation mirrored E2FB expression, unlike CDKA;1 .

3. Phenotypic Observations:

   • Disrupted filamentous cell structures under auxin-free conditions.

   • Increased mitotic figures in arrested cells .

Technical Considerations

• Cross-Reactivity: No cross-reactivity observed with other E2F family members .

• Limitations: Requires denaturing conditions for optimal detection in immunoblots.

• Alternative Methods: Used in conjunction with p13^suc1-affinity binding and histone H1 kinase assays for comprehensive cell cycle analysis .