JCS03292 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. The I-domain cannot bind the B- or G-domain of nucleostemin. (A) Diagram of nucleostemin mutants used to distinguish between a nucleoplasmic-retaining and a NoLS-masking mechanism of the I-domain. (B) Physical interactions between the I-domain and different parts of nucleostemin were examined by affinity binding assays. GST fusions of the I-domain (2 μg) failed to bind the B- and/or G-domain-containing mutant proteins, regardless of their GTP-binding state. Homodimerization of another new nucleolar factor was included as the positive control (+).

• Supplemental Figure 2 -

  Fig. S2. Colocalization of nucleostemin and RSL1D1 within the nucleolus. (A-C) Colocalization of HA-tagged nucleostemin (red) and GFP-tagged RSL1D1 (green), B23 (red) and RSL1D1-gfp (green), and fibrillarin (red, Fib) and RSL1D1-gfp (green) determined by double-labeled immunofluorescence and confocal analyses in A, B and C, respectively. High magnifications of the indicated areas (squares) is shown in A2, B2 and C2. Colocalization is quantified in A3, B3 and C3 where all pixels were plotted on the basis of their red (x-axis) and green (y-axis) fluorescence intensities, and pseudocolored on the basis of the event frequency. Dashed lines delineate the nucleo-cytoplasmic boundaries. Bars, 5 μm for A1, B1, and C1; 2 μm for A2, B2, C2.