Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport.

Verification of RNF38 substrates by reconstituted ubiquitination assays in vitro and in the cell Since the OUT screen revealed the association of RNF38 substrates with intracellular and nuclear transport pathways, we chose to verify RNF38-catalyzed ubiquitination of Ran and RanGAP1, two key nuclear transport proteins ( 53 ) with highly significant -Log 10 p values of 1.28 and 1.76, respectively ( Fig. 3 , D and E ).

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