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. 2020 Jun 19;11:3106. doi: 10.1038/s41467-020-16939-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a IPs of FLAG-UPF1: either WT or one of its variants. HEK293T cells transiently expressing the indicated protein were treated with either DMSO or MG132 for 12 h before harvesting. The cell extracts were treated with RNase A and then subjected to IPs with the α-FLAG antibody; n = 3. b Quantitation of the Western blotting images presented in (a). Signal intensities of the Western blot bands were quantified. The levels of hyperphosphorylated UPF1 and coimmunopurified cellular proteins were normalized to the amounts of immunopurified FLAG-UPF1. The normalized levels obtained in the IP of FLAG-UPF1-WT were arbitrarily set to 1.0. Data are presented as mean values ± standard deviations (SD) and statistical significance; Two-tailed, equal-sample variance Student’s t test was carried out to calculate the P values. *P = 0.0102 and **P < 0.0039. The exact P values are provided in a Source Data File; n = 3. c IPs of FLAG-CTIF in cells depleted of UPF1. HEK293T cells either depleted or not depleted of endogenous UPF1 were transiently transfected with a plasmid expressing FLAG-CTIF. The cells were treated with MG132 for 12 h before harvesting. The extracts of the cells were treated with RNase A and then subjected to IPs using the α-FLAG antibody; Two-tailed, equal-sample variance Student’s t test was carried out to calculate the P values. *P = 0.0241 (eEF1A1) and 0.0184 (DCTN1); n = 3. d IPs of FLAG-UPF1-WT or its variants in the extracts of cells treated with puromycin. HEK293T cells transiently expressing FLAG-UPF1-WT or one of its variants were treated with MG132 for 12 h and puromycin for 1 h before cell harvesting. The RNase A–treated extracts of the cells were subjected to IPs with the α-FLAG antibody. The immunoglobulin heavy chain detected after IPs is denoted by an arrowhead; n = 2. e IPs of FLAG-UPF1-HP using the extracts of cells depleted of eEF1A1. As performed in panel d, except that HEK293T cells either depleted or not depleted of endogenous eEF1A1 were transiently transfected with a plasmid expressing FLAG-UPF1-HP; n = 2. f IPs of FLAG-CTIF in the extracts of cells depleted of UPF1. As performed in panel d, except that HEK293T cells either depleted or not depleted of endogenous UPF1 were transiently transfected with a plasmid expressing FLAG-CTIF; n = 2; Source data are provided as a Source Data File.