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. 2011 Jun 30;39(18):8105–8121. doi: 10.1093/nar/gkr508

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — The recruitment of Imp4 to early pre-ribosomes is secondary to the assembly of UTP-B/U3. (A, C and E) Sedimentation behavior in sucrose gradients of Imp4 (A and C) and Pwp2 (E) in the presence and absence of Nan1 (A), Pwp2 (C) and Imp4 (E). Cellular extracts prepared from the indicated yeast strains (top) grown in medium containing either galactose (left panels) or glucose (right panels) were resolved in 7–50% linear sucrose gradients. After ultracentrifugation, 0.5 ml fractions were collected from the top of the tube. The content of Imp4–MYC (A and C) and Pwp2–MYC (E) in each fraction was analyzed by anti-MYC immunoblotting (first panels from top). In parallel, total RNAs were prepared from each fraction and analyzed by northern blot as indicated in Figure 2. The number of each fraction and the sedimentation points of 40S and 80S particles are indicated at the bottom. Gal, galactose; Glu, glucose; WB, western blot; NB, northern blot. (B, D and F) Co-immunoprecipitation of Imp4 (B and D) and Pwp2 (F) with pre-RNAs and U3 snoRNA in the presence and absence of Nan1 (B), Pwp2 (D) and Imp4 (F). Total cellular lysates (lanes 1–4) and anti-MYC immunoprecipitates (lanes 5–8) prepared from the indicated yeast strains and growth conditions (top) were analyzed by anti-MYC and anti-HA immunoblotting (upper panels), and northern blot analysis as described in Figure 2. TCL, total cellular lysates; IP, immunoprecipitation.