Fig. 4.

The USP domain expressed in trans stabilizes VP1-2 C65A mutants. (a) Variant encoding approximately 60% of the protein NT6 was expressed in comparison to NT3, each containing C65 or C65A as indicated. As for NT3, substitution of C65A in NT6 resulted in a profound decrease in levels of the protein (cf. lanes 4 and 5) which could be stabilized by proteosome inhibition (cf. lanes 5 and 6). (b) Cells were transfected with constructs for constitutive expression of NT3.C65A, NT6, or NT6.C65A as indicated, together with the vector for the Dox-inducible expression of NT1. Twenty-four hours after transfection, Dox was added to one set of duplicate transfections and cells were incubated for a further 24 h. Samples were then probed simultaneously for expression of the various species. Dox induction resulted in expression of NT1 and a concomitant increase in NT3.C65A and NT6.C65A. An increase was seen also for NT6 itself.