FIG 3.

LGP2 interacts with MDA5 and activates RIG-I signaling and NF-κB signaling. (A) PAMs were mock infected or infected with PRRSV (MOI = 1) for 12 and 24 h, and cell lysates were immunoprecipitated with an LGP2 antibody. Immunoblots of MDA5, RIG-I, and LGP2 are shown. GAPDH is shown as an internal control. (B) PAMs were mock infected or infected with PRRSV (MOI = 1) for 24 h. Cell lysates were immunoprecipitated with the RIG-I antibody, and immunoblots were shown to detect the interaction between LGP2 and RIG-I. (C) PAMs were mock infected or infected with PRRSV (MOI = 1) for 24 h. Cell lysates were immunoprecipitated with MDA5 antibody, and immunoblots showed the interaction change between LGP2 and MDA5. (D) Marc-145 cells were cotransfected with Myc-tagged LGP2 and Flag-tagged MDA5 plasmids for 24 h, and cells were mock infected or infected with PRRSV (MOI = 1) for 24 h. Cell lysates were immunoprecipitated with Myc antibody, precipitated with Flag-tagged MDA5, and Myc-tagged LGP2 were detected using immunoblotting. (E) Marc-145 cells were transfected with empty vector and LGP2 plasmids for 24 h and incubated with PRRSV at 0, 12, 24, 36, and 48 hpi, cells were collected. The protein levels of p-IRF3, IRF3, and PRRSV N were determined using Western blot analysis. (F) Marc-145 cells were treated as described in panel E. Cells were collected, and nuclear and cytoplasmic protein samples were extracted using a nuclear and cytoplasmic protein extraction kit. Western blotting was performed to detect the expression of NF-κB p65, RIG-I, MDA5, and PRRSV N. Histone H3 is used as an internal nuclear control, and GAPDH serves as the cytoplasmic internal control. (G) PAMs were transfected with scrambled siRNA or LGP2 siRNA (si-3) for 24 h and then infected with PRRSV (MOI = 1) for 0, 6, and 12 h. The protein expression of PRRSV N, p-IRF3, and IRF3 are shown, as detected by Western blotting. For the Western blot, the relative band density was normalized to the loading control GAPDH and then compared to the corresponding control.