Fig. 8.

p32 is present in the Nop52-associated pre-60S particles. A, The nuclear fraction prepared from control cells (control) that stably expressed hygromycin-resistant protein constructed by the transfection of pcDNA5/FRT/TO vector into Flp-In T-REx 293 cells or the inducible FLAG-Nop52 expression cells (FLAG-Nop52) was separated into 20 fractions by sucrose gradient ultracentrifugation. The absorbance at 254 nm was monitored to generate the profile indicated. The pre-60S fractions 11–15 were combined for each of the profiles independently and used for immunoprecipitation with anti-FLAG-fixed beads. B, The isolated proteins were analyzed by 10% SDS-PAGE and visualized by silver staining. Asterisk indicates FLAG-Nop52 as bait. The molecular masses (kDa) are indicated at the left. C, Association of p32 with Nop52 in isolated pre-60S ribosomal particles was confirmed by immunoblot (IB) analysis with anti-p32. An aliquot (5%) of the pre-60S combined fractions prepared from control or FLAG-Nop52 was used as input. IP: Immunoprecipitation.