Fig. 3.

Zein–Nef is rapidly degraded. Protoplasts isolated from young leaves of transgenic tobacco expressing zein–Nef (B, C, and zein–Nef in A) or transformed with the empty vector pGA470 (control in A) were subjected to pulse-labelling with [³⁵S]Met and [³⁵S]Cys for 1 h followed by chase for the indicated times. Proteins were immunoprecipitated with anti-Nef antibodies from homogenates prepared as described below. (A) Protoplasts were homogenated with reducing buffer. (B) Protoplasts were homogenated with reducing buffer (soluble). The insoluble material of the first homogenation was solubilized with denaturating buffer (insoluble). As a control, protoplasts were also directly homogenized with denaturing buffer (denat.). (C) Pulse–chase was performed using protoplasts treated (+) or untreated (–) with brefeldin A (BFA). Both protoplasts and their incubation media were collected and homogenated with reducing buffer. Analysis was by SDS–PAGE and fluorography. In each panel, numbers on the left indicate the positions of molecular mass markers, in kilodaltons.