FIGURE 1.

TRN2 regulates the nuclear localization of HuR. A and B, TRN2 siRNA reduces the level of TRN2 RNA. HeLa cells were transfected with either an siRNA that targets TRN2 (siTRN2) or control siRNA (siC). Their total RNA content was isolated and analyzed by slot blotting, probing for TRN2 and 18 S (loading control) RNAs. Quantification was performed using ImageQuant software, and average values of TRN2 RNA levels, relative to 18 S RNA levels, are graphed in B, with error bars representing S.E. of four independent experiments. The asterisk indicates that the observed difference is significant, with p = 0.0006. C–E, knockdown of TRN2 causes HuR to accumulate in the cytoplasm. Cells were treated as described for A. They were fixed, and immunofluorescence was performed by staining with anti-HuR antibody and DAPI (C). Scale bars = 20 μm. Alternatively, the total content of lysed cells was separated into nuclear (N) and cytoplasmic (C) fractions and analyzed by Western blotting (D) with antibodies against HuR, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1; nuclear marker), and α-tubulin (cytoplasmic marker). Quantification of HuR and α-tubulin band intensities was determined using ImageQuant software, and average values of cytoplasmic HuR levels relative to α-tubulin are graphed in E, with error bars representing S.E. of three independent experiments. The asterisk indicates that the observed difference is significant, with p = 0.0053. All images shown in C and the blot in D are representative of three independent experiments.