FIG. 5.

Npa2p is present in large complexes, most likely pre-60S r-particles. Total cell extracts were obtained from IVY317 (Npa2p-TAP, Has1p, Rpl3p), IVY325 (Npa1p-HA), IVY347 (HA-Dbp6p), IVY411 (Nop8p-HA), and IVY409 (Rsa3p-HA) cells following growth at 30°C. About 15 A₂₅₄ units of cell extract were resolved in 7% to 50% sucrose gradients containing a low concentration of Mg²⁺ to dissociate ribosomes into subunits. Sedimentation is shown from left to right. The sedimentation positions of 40S and 60S ribosomal subunits are indicated. (A) Fractions were collected from the gradients, and proteins were extracted from the same volume of each fraction, resolved on 7% SDS-PAGE gels, and subjected to Western blotting. The blots were decorated with specific antibodies detecting the proteins indicated. (B) RNA was extracted from equal volumes of each fraction, resolved on a 1.2% agarose-formaldehyde gel, and transferred onto a nylon membrane. The same membrane was hybridized with different probes. Probe names are indicated between parentheses. T stands for total extract, and numbers correspond to fraction numbers.