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Updated: Jul 15, 2026

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Regulation of Cited2 expression provides a functional link between translational and transcriptional responses during
Twan van den Beucken1, Michael G Magagnin, Kim Savelkouls
1Department of Radiation Oncology, Maastricht University, Maastricht, The Netherlands.
Background And Purpose:
Protein synthesis rates are greatly reduced under hypoxic conditions as a consequence of an overall inhibition of mRNA translation. Certain specific mRNA species have the ability to escape this general translational repression. At the cellular level this results in differential protein expression during hypoxic conditions. The objective of this study was to characterize the translational regulation of the postulated HIF-1alpha antagonist Cited2.
Materials And Methods:
DU145 prostate carcinoma cells and mouse embryonic fibroblasts with a homozygous knock-in mutation for eIF2alpha (S51A) or wild-type eIF2alpha were exposed to severe hypoxia after which both total mRNA and efficiently translated mRNA were isolated. Quantitative RT-PCR was used to measure and compare changes in transcription (total mRNA) with changes in translation (efficiently translated mRNA fraction).
Results:
We show using HIF-1alpha null MEF cells that transcriptional induction of Cited2 during hypoxia is dependent on HIF-1alpha. Although global mRNA translation is inhibited during hypoxia Cited2 mRNA remains efficiently translated. An evolutionary conserved upstream open reading frame (uORF) in the 5'UTR of Cited2 did not stimulate translation in an eIF2alpha dependent manner during hypoxia.
Conclusions:
Selective translation Cited2 by an eIF2alpha independent mechanism establishes a link between translation and HIF-1 dependent transcription during hypoxia.
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