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Fig. 3. Overexpression of GNL3L brings ERR
and ERR
into the nucleolus. (A) To generate a nucleolar form of GNL3L (NoG3l), we replaced the N-terminal nucleolus-targeting domain of GNL3L with the corresponding region of nucleostemin (grey bar), which has a stronger nucleolus-targeting activity than GNL3L but lacks the ability to bind ERR. To create a nucleoplasmic form of GNL3L (nls-I), we fused the I-domain of GNL3L to an SV40 nuclear localization signal (oval). (B) Affinity-binding assays show that both nls-I and NoG3l maintain the ability to bind ERR. To measure the effect of GNL3L overexpression on the distribution of ERR, U2OS cells were transfected with Myc-tagged ERR alone (C1), Myc-tagged ERR and HA-tagged wild-type GNL3 (C2) or mutant GNL3L (C3,C4) or HA-tagged GNL3L constructs alone (C5 and C6). Double-transfected cells were labeled with anti-Myc (red) and anti-HA (green) antibodies, and visualized by confocal analyses. Single-transfected cells were immunostained with anti-fibrillarin antibody (Fib) and anti-Myc or anti-HA antibody. The ERR (red) fluorescence intensities are measured quantitatively along the lines indicated by arrows, shown in the right panels of (C1'-C3'), and the nucleolar regions (No) are indicated by the increase of green fluorescence. Compared to cells transfected with only ERR, the fluorescence intensity of ERR in the nucleolus is increased in cells cotransfected with NoG3l or the wild-type GNL3L. By contrast, the nls-I mutant does not change the distribution of ERR (C4). Neither does ERR alter the distribution of GNL3L (C5) or NoG3l (C6). The same analyses were performed using ERR (D1-D3) and ERR
(E1-E3). Our results showed that, when coexpressed with wild-type GNL3L (D2) or NoG3l (D3), ERR begins to accumulate in the nucleolus. GNL3L overexpression has little or no effect on the distribution of ERR (E2,E3). Bars, 10 µm.
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