Figure 4. CypA interacts with RIG-I and MAVS to activate RIG-I signaling pathway.

(A–C) Luciferase activity of lysates in 293T/CypA- cells transfected for 24 hr with luciferase reporter constructs IFN-β-Luc (A), ISRE-Luc (B) or NF-κB-Luc (C), plus Flag-MAVS, Flag-RIG-I-N, Flag-MDA5-N, Flag-IRF3/5, or Myc-TBK1, along with Myc-CypA or an empty vector. Results are presented relative to the luciferase activity in control cells treated with luciferase reporter and empty vector. (D) Native PAGE and immunoblot analysis of IRF3 in dimer or monomer form and phosphorylated IRF3 in 293T/CypA- cells transfected for 24 hr with Flag-MAVS, Flag-RIG-I, Flag-MDA5, or Myc-TBK1, along with an empty vector or Myc-CypA. (E) Immunoblot analysis of lysates of 293T/CypA+ cells transfected for 24 hr with Flag-MAVS, Flag-RIG-I, Flag-MDA5, or Flag-IRF3, along with Myc-CypA, followed by immunoprecipitation with anti-Flag beads. (F and G) Immunoblot analysis of lysates in WT BMDMs infected with SeV for 6 hr, followed by immunoprecipitation with control mouse IgG or anti-CypA antibodies. Lysates and immunoprecipitation extracts were probed with CypA and RIG-I (F) or MAVS (G) antibodies. (H) Confocal microscopy of endogenous CypA and MAVS or RIG-I in 293T/CypA+ cells, treated with SeV for 6 hr. Scale bars, 10 μm. 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.24425.014