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Fig. 4. NRAGE induces ubiquitin-dependent Che-1 degradation. (A) Three representative sections showing endogenous Che-1 in SY5Y cells transfected with expression vector expressing EGFP-NRAGE fusion protein, subjected to indirect immunofluorescence analysis as described in Materials and Methods. In the merged image, no fluorescence signal derived from Che-1 (red) was detectable in cells overexpressing EGFP-NRAGE (green). (B) Western blot analysis of HeLa cells transfected with either Myc-NRAGE or with empty control vector, harvested at the indicated times and analysed on SDS-PAGE. The immunoreactivity of Che-1 and Myc-NRAGE proteins was detected by probing blots with anti-Che-1 and anti-Myc antibodies, respectively. Samples were normalised for protein content by reprobing blots with anti -tubulin. (C) In vivo ubiquitylation assay was performed with HeLa cells transiently co-transfected with plasmids expressing HA-tagged ubiquitin and Myc-NRAGE. After transfection, cells were treated without or with 20 µM MG132 for 4 hours and subjected to immunoprecipitation (IP) and western blotting (WB) analysis with the indicated antibodies. Che-1-ubiquitin conjugates were detected by using anti-HA monoclonal antibody.

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