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. 2017 Jun 8;6:e24425. doi: 10.7554/eLife.24425

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© 2017, Liu et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 7. — (A) Luciferase activity of lysates in 293T/CypA- cells transfected for 24 hr with IFN-β-Luc and Flag-TRIM25, Flag-Smurf1, Flag-AIP4, Flag-RNF125, or Flag-RNF5, along with CypA or an empty vector and then treated with SeV for 6 hr. Results are presented relative to the luciferase activity in control cells transfected with luciferase reporter and empty vector. (B) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids for 24 hr. (C) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids for 24 hr, followed by immunoprecipitation with anti-MAVS antibody. (D) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids for 24 hr, followed by immunoprecipitation with anti-Flag beads. (E) Immunoblot analysis of lysates in WT and Ppia^−/− BMDMs infected with SeV for 6 hr, followed by immunoprecipitation with control mouse IgG or anti-MAVS antibodies. Lysates and immunoprecipitation extracts were probed with K48-Ub, MAVS and CypA antibodies. (F) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids, followed by immunoprecipitation with anti-Flag beads. (G and H) Immunoblot analysis of lysates in 293T/CypA+ cells transfected for 24 hr with Flag-CypA (G) or Flag-TRIM25 (H), along with HA-tagged deletion constructs of MAVS, followed by immunoprecipitation with anti-Flag beads. (I) Immunoblot analysis of lysates in 293T/CypA+ cells transfected with HA-Ub, plus Myc-MAVS, Myc-MAVS KK-AA (K371A plus K420A), or the double point substitution construct Myc-MAVS KK-RR (K7R plus K10R), along with Flag-TRIM25 or an empty vector, followed by immunoprecipitation with anti-Myc beads. (J) Quantitative PCR analysis of IFNB1 mRNA in 293T/RIG-I^−/− cells pretreated for 1 hr with BAY 11–7082 (5 μM) or DMSO, and then transfected for 48 hr with Flag-RIG-I, Flag-MAVS or an empty vector, along with scrambled siRNA or CypA siRNA (top). The phosphorylated IRF3 and p65 were detected by immunoblot (below). (K) Quantitative PCR analysis of SeV M mRNA in 293T/RIG-I^−/− cells pretreated for 1 hr with BAY 11–7082 (5 μM) or DMSO, then transfected for 48 hr with Flag-RIG-I, Flag-MAVS or an empty vector, along with scrambled siRNA or CypA siRNA, and then infected with SeV for 6 hr. Results are presented relative to mRNA level of SeV M in control cells transfected with empty vector and infected with SeV. Data are shown as mean ± SD (n = 3). *p<0.05 and **p<0.01 (unpaired, two-tailed Student’s t-test). Data are representative of at least three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.24425.018

Figure 7—source data 1. Quantification of luciferase activity, IFN-β production and SeV replication for Figure 7.
DOI: http://dx.doi.org/10.7554/eLife.24425.019

elife-24425-fig7-data1.xls^{(62.5KB, xls)}

DOI: 10.7554/eLife.24425.019