In vitro binding of MBP-Nmd3 to 60S but not 80S ribosomes. (A) Increasing amounts of MBP-Nmd3 were incubated with Rpl25-myc–containing 60S subunits and immunoprecipitated with anti-myc antibody and protein A beads. Bound proteins were eluted in Laemmli sample buffer, separated on 12% SDS-PAGE, and stained with Coomassie brilliant blue. See Materials and methods for details. Lane 1 shows MBP-Nmd3 without 60S; lanes 2–7 show 60S-myc with increasing amounts of MBP-Nmd3 as indicated. The molar ratio of Nmd3 to 60S subunits is given for the input and bound samples. (B) Nmd3 does not bind 80S ribosomes. MBP-Nmd3 was incubated alone (lanes 1 and 2), with 60S (lanes 3 and 4), or with 80S subunits (lanes 5 and 6). Samples were layered over 60% sucrose cushions and centrifuged. Supernatants (S) and pellets (P) were separated on a 12% SDS-PAGE, and proteins were visualized by Coomassie staining. (A and B) The positions of molecular mass markers (M) are given in kilodaltons.