Figure 2.

Heat shock induces cytoplasmic relocalization of HuR and poly(A)⁺ RNA sequestration in the nucleus. (1–12) Localization of HuR, hnRNP D, hnRNP A1, La protein, and ribosomal RNA before and after heat shock. HeLa cells were grown on coverslips, heat shocked, fixed, permeabilized, and immunofluorescently stained as described in Materials and Methods. In 1–6, the cells were singly or doubly stained with 3A2 and/or polyclonal anti-hnRNP D and detected by using fluorescein isothiocyanate- or rhodamine-labeled secondary antibody, respectively. (13–15) The distribution of poly(A)⁺ RNA was examined in HeLa cells before and after heat shock by using 5 ng/μl digoxigenin-labeled oligo(dT₃₅) probe (13, 14) and a 1/200 dilution of sheep antidigoxigenin Fab-rhodamine antibody (Boehringer). Cells in 15 were treated the same way as those in 13 and 14, except that during the hybridization step they were incubated without the digoxigenin-labeled oligo(dT₃₅) probe. All images were captured by using the Bio-Rad MRC-1024 confocal microscope.