Fig. 2.

The USP domain can deubiquitinate itself. (a) Cell lines containing inducible NT1 (lanes 1 to 4) and NT1.C65A (lanes 5 to 8) were established and accumulation measured after Dox induction with or without the addition of MG132 (added for 4 h before harvesting) as indicated. The species NT1¹ and NT1² were readily observed for NT1.C65A and could be seen for NT1 (Fig. 1). NT1* was specific for NT1.C65A and its accumulation was significantly increased by MG132 addition. (b) The same samples as in panel a were probed for the presence of total ubiquitinated species using the anti-ubiquitin antibody FK2. We did not observe major changes in the total ubiquitinated species upon NT1 (or NT1.C65A) induction. However, MG132 treatment resulted in the loss of free ubiquitin and a general increase in higher-molecular-weight species. A novel ubiquitinated species appeared upon Dox induction of NT1.C65A. This species labeled Ub1 (lane 8) was not observed for NT1 induction. (c) Alignment of the same blots as in panels a and b. The Dox-induced NT1.C65A band NT1* (lane 1) comigrates with the novel Dox-induced ubiquitinylated band, Ub1 (lane 3).