Fig. 1. Immunofluorescence reveals components involved in translation and NMD are found near nascent transcripts in HeLa nuclei. (A) Some UPF1, 2 and 3 is nuclear. Cells were fixed, UPF1, 2, or 3 indirectly immunolabelled with Cy3, and equatorial sections through nuclei collected using a confocal microscope. (Rows 1-3) Fraction cellular intensity found in nuclei for cells grown with or without leptomycin B or actinomycin D. *Difference relative to value in row 1, significant at 99.999% level (Student's t-test). Although the nuclear signal given by UPF1 and 2 is faint, quantitative analysis reveals it constitutes a significant fraction of the total. Scale bar: 10 µm. (B) Anti-Br binds to Br-RNA (left), but not when blocked by anti-X bound to its target (right). (C) Some antibodies block access of anti-Br to nascent Br-RNA. HeLa cells were grown briefly in Br-U to label nascent transcripts, and fixed; then the resulting nascent Br-RNA was indirectly immunolabelled with Cy3, and single equatorial optical sections through nuclei collected using a confocal microscope. In the absence of a blocking antibody, nuclear fluorescence marks nascent Br-RNA (left), which is reduced by co-incubation with anti-UPF2 (right); other antibodies have intermediate effects (middle). Scale bar: 20 µm. (D) Colocalization revealed by antibody blocking: various antibodies prevent access of anti-Br to Br-RNA (left), and vice versa (right). Fluorescence intensities over >300 nuclei in images like those in the right-hand panel in (C) were expressed relative to those in the left-hand panel. *Difference relative to value in row 1, significant at 99.999% level (Student's t test).