FIGURE 1.

In vitro sumoylation of keratins and vimentin. A, Coomassie stain of purified recombinant human K8, K18, K5, K14, and vimentin (Vim) resolved on a 4–20% gradient SDS-PAGE gel. B and C, purified keratins, vimentin, or no substrate (−) were incubated with E1/E2 SUMO ligases, ATP, and either SUMO-1, SUMO-2, or SUMO-3. The resulting reaction products were analyzed by immunoblotting for the ATP-dependent formation of SUMO-1 and SUMO-2/3-containing intermediate filament conjugates. The asterisks denote SUMO monomers (*) or dimers (**). Keratin and vimentin blots serve as loading controls.