Figure 2.

 The Upf1p is associated with eRF3 [PSI⁺] aggregates. Cytoplasmic extracts from isogenic [PSI⁺] and [psi⁻] variants of strain 7G-H66 upf1Δ and containing FLAG–UPF1 inserted into a centromere plasmid were fractionated by centrifugation through a sucrose cushion as described previously (Paushkin et al. 1997b). Supernatant (cytosol), sucrose pad (sucrose), and pellet fractions were subjected to SDS–PAGE, and the distribution of eRF1, eRF3, and Upf1p within these fractions was determined by immunoblotting with polyclonal antibody against eRF1 and eRF3 and a monoclonal antibody against the Flag epitope. A 95-kD protein cross-reacts with anti-flag antibody in strain 7G-H66 and has the same distribution in [PSI⁺] and [psi⁻] cells. This 95-kD protein is not present in extracts prepared from strain BJ3505 (see Fig. 1).