Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1α and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1α containing plasmid. A mutant HIF-1α incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1α-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications. J. Cell. Biochem. 115: 271–280, 2014. © 2013 Wiley Periodicals, Inc.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.