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The Dark Side of EGFP: Defective Polyubiquitination 1 1 1 1 2 2 3 1 Mathijs Baens *, Heidi Noels , Vicky Broeckx , Sofie Hagens , Sabine Fevery , An D. Billiau , Hugo Vankelecom , Peter Marynen 1 Applied Human Genomics, Center for Human Genetics, Molecular Genetics - Flanders Interuniversity Institute for Biotechnology (VIB), University of Leuven, Leuven, Belgium, 2 Laboratory of Experimental Transplantation, University of Leuven, Leuven, Belgium, 3 Laboratory of Cell Pharmacology, University of Leuven, Leuven, Belgium Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-kB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered. Citation: Baens M, Noels H, Broeckx V, Hagens S, Fevery S, et al (2006) The Dark Side of EGFP: Defective Polyubiquitination. PLoS ONE 1(1): e54. doi:10.1371/journal.pone.0000054 INTRODUCTION in a number of experiments. To our surprise we observed that The extensive use of EGFP as a live cell reporter is