Fig. 2

NMD factors UPF1 and UPF3 localise to neurites while protein levels are downregulated during neuronal differentiation. a. Neural stem cells were differentiated by the addition of forskolin and retinoic acid. Protein samples were prepared before (day 0 (d 0)) and at the indicated time (d, days) after induction of differentiation. Equal amounts of protein were subjected to 8 % SDS PAGE and analysed for β-III Tubulin, UPF3B and UPF1 protein levels by Western blotting. Samples were probed for GAPDH protein as loading control. Proteins with faster mobility indicated by asterisk reflect the behaviour of UPF1 and UPF3B and are likely to be alternate isoforms. The relative changes of UPF3B and UPF1 protein levels were quantitated using GAPDH as reference, with levels at day 0 defined as 1. The mobility of relevant molecular mass markers is indicated. b. UPF1 and UPF3B protein localisation in undifferentiated HCN-A94 cells. Cells were stained with DAPI (blue) and with either anti-UPF3B antibody (red) or anti-UPF1 antibody (green) were analysed by confocal microscopy. Shown are separate channels and merged images from a single section. c. UPF3B and UPF1 localisation in neurons. Neural stem cells differentiated for 8 days and then stained with DAPI and either anti-UPF3B antibody or anti-UPF1 antibody were analysed by confocal microscopy. Shown are separate channels and merged images from a single section through the nucleus and 3 days renderings of corresponding Z-stacks. The scale bars represent 20 μm. Note that UPF3B and UPF1 are detected in neurites and UPF1, but not UPF3B, largely is excluded from the nucleus. d. Nucleolar localisation of UPF3B. Undifferentiated or for 8 day differentiated neural stem cells were stained with anti-UPF3B antibodies, anti-Fibrillarin antibodies and DAPI and then analysed by confocal microscopy. Sections shown focus on the nuclear compartment. The scale bars represent 20 μm. Note that UPF3B is enriched in nucleoli stained by anti-Fibrillarin antibodies