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. 2006 May 5;34(8):2355–2363. doi: 10.1093/nar/gkl277

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© The Author 2006. Published by Oxford University Press. All rights reserved

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Increasing the length of the TPO uORF results in a hUpf1-independent decline of mRNA steady-state levels. (A) Northern blot analysis of total cytoplasmic RNA from HeLa cells that were transfected with siRNAs directed against hUpf1 to specifically deplete endogenous hUpf1 protein (lanes 5–8, 11, 12). Cells transfected with siRNAs directed against luciferase (lanes 1–4, 9, 10) served as negative controls. After 20 h the siRNA treatment, cells were cotransfected with TPO minigene constructs wt (lanes 1 and 5), ΔIntron 4 (lanes 2 and 6), ΔG (lanes 3 and 7), NS40 ΔG (lanes 4 and 8), β-globin wt (lanes 9 and 11) and NS 39 (lanes 10 and 12) together with the control plasmid. Percentages are means that were calculated from three independent experiments. (B) Immunoblot analysis of HeLa cell extracts transfected with hUpf1 siRNA. Immunoblotting was performed with a hUpf1-specific antibody (lower panel). The membrane was reprobed with a tubulin-specific antibody (upper panel) to control for equal loading.