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New Fusion Trans-Activator Plasmids for Studying Signal Transduction,,,Pathways

tion sites for insertion of the transcription factor activation domain sequence, (2) expression of the fusion trans-activator protein driven by the CMV promoter, a strong promoter that allows high-level constitutive expression in a variety of cell lines and (3) the neomycin-resistance gene, which facilitates selection of stable cell lines that express the fusion trans-activator protein of interest.

The backbone of the pFA-CMV plasmid was derived from Stratagenes pcmv-script vector.7 This backbone, along with a polylinker encoding 10 unique cloning sites, was fused to the DNA binding domain of GAL4. To test the functionality of the pFA-CMV plasmid, we cloned several activation domains, including the CREB, c-Jun and Elk1 domains, into the multiple cloning site of the pFA-CMV plasmid. Expression of the fusion proteins was confirmed by Western blot analysis with an antibody to the GAL4 DNA binding domain. All constructs were tested and confirmed for specificity and activation levels in transient transfection assays in Chinese hamster ovary (CHO) and HeLa cells (unpublished data). We also cloned the activation domains of the ATF2 and c-Fos transcription factors into the pFA-CMV plasmid, creating two new fusion trans-activator plasmids.

Design of the pFA-ATF2 and pFA-cFos Fusion Trans-Activator Plasmids

figure 3A

c-Fos is a proto-oncogene and an immediate-early gene.8-10 Although the direct activator of c-Fos remains somewhat elusive, c-Fos is thought to be activated by Fos-regulating kinase (FRK), which is distinct from JNK and extracellular signal-regulated kinase (ERK). A portion of c-Fos (amino acid residues 208 to 313) that contains the FRK phosphorylation site (Thr-232) has been shown to be
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Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. Stable HeLa Luciferase Reporter Cell Lines Expressing GAL4 Fusion Transactivators
3. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
4. Comparison of Performance between the AlphaQuest - HTS and the Fusion α Multilabel Reader for Detection of cAMP
5. Mobile Phase Optimization for the Analysis of an Antibody-Based Fusion Protein and Aggregates using a TSKgel Super SW3000 Size-Exclusion Column
6. New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction Pathways
7. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
8. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
9. Enzyme Immunoassay for Studying Intracellular Levels of cAMP
10. Assess the In Vivo Activation of Signal Transduction Pathways with PathDetect Reporting Systems
11. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
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