Figure 6.

Mdn1 Is Required for the Assembly of the Nucleolar Nsa1 Particles

(A and B) Rix7-GFP, Ppp1-GFP, Ytm1-GFP, and Nsa1-GFP signals in wild-type cells with or without Rbin-1 (1 μM, 1 hr at 25°C) treatment were examined. Representative images are shown in (A). Scale bar, 2 μm. The intensity of GFP signals in the nucleolus (NL) divided by that in nucleoplasm (NP) is shown in (B). Mean ± SEM, n = 30 cells.

(C and D) Rix7-GFP signals were examined in asynchronous cells (0 min), 1 μM Rbin-1-treated cells (30–120 min), and cells after washing out Rbin-1 (wo-5-60 min) at 29°C. Representative images are shown in (C). Scale bars, 2 μm. The intensity of the Rix7-GFP signal in the nucleolus (NL) divided by that in the nucleoplasm (NP) is shown in (D). Average is indicated by black bar (mean ± SEM, n = 30 cells). Asterisks indicate unpaired two-tailed Student’s t test significance value: ^∗∗∗p < 0.001.

(E–I) Whole-cell extracts (Input) were prepared from wild-type or mdn1-F1093L cells treated with Rbin-1 (1 μM) for indicated time. Nsa1-5FLAG was immunoprecipitated with anti-FLAG antibodies (IP: FLAG). The indicated proteins ([E] Rix7-GFP, [F] Ppp1-GFP, [G] Ytm1-3HA, [H] Rix7-GFP, and [I] GFP-Mdn1) and Nsa1-5FLAG were analyzed by SDS-PAGE and western blotting. The grouping of images from different parts of the same gel is indicated by dividing lines. The graph in (H) shows relative amount of Rix7 in IP samples (mean ± range, n = 2).

See also Figure S7.