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Fig. 3. Nucleostemin interacts with RSL1D1. (A) Protein sequences of mouse RSL1D1 (GenBank accession no. NM_025546) and two closely related genes, L10A (GenBank accession no. NM_011287), and L10 (GenBank accession no. XM_138143), were aligned by the Clustal W (1.81) program. Shaded and underlined areas represent the ribosomal L1 and the coiled-coil domain, respectively. Three putative NLS are marked in bold. ^*, fully conserved residues;:, conservation of strong groups;., conservation of weak groups. Biochemical interaction between nucleostemin and RSL1D1 was shown by affinity binding assays using a GST-RSL1D1 fusion protein to pull down HA-tagged nucleostemin (B1) or a GST-nucleostemin fusion protein to pull down HA-tagged RSL1D1 (B2). (C) The nucleostemin-RSL1D1 interaction was confirmed by coimmunoprecipitation in both directions. HEK293 cells were co-transfected with HA-tagged nucleostemin and Myc-tagged RSL1D1 and immunoprecipitated with anti-Myc antibody (rows 1 and 2, left column, ${alpha}$ -myc), anti-HA antibody (rows 3 and 4, rows, left column, ${alpha}$ -HA), or mouse IgG (rows 1-4, right column). The co-purified proteins (rows 1 and 3) and the immunoprecipitates (rows 2 and 4) were detected by immunoblotting with the indicated polyclonal antibodies. (D) Left panel: Myc-tagged RSL1D1 was co-purified with endogenous nucleostemin in HEK293 cells by nucleostemin antiserum ( ${alpha}$ -NS), but not by preimmune serum (Cntrl). Right panel: endogenous nucleostemin was also be co-purified with Myc-tagged RSL1D1 by anti-Myc antibody ( ${alpha}$ -myc) but not by mouse IgG.

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