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. 2019 Aug 28;93(18):e00322-19. doi: 10.1128/JVI.00322-19

FIG 1.

FIG 1

NDV infection induced MAVS degradation. (A) HeLa cells were mock treated or infected with NDV at an MOI of 1. Cells were harvested at 6, 12, 18, and 24 hpi and detected using immunoblot (IB) analysis with anti-MAVS, anti-NP, or anti-β-actin antibody. (B) HeLa cells were mock treated or infected with NDV at an MOI of 0.01, 0.1, 1, or 5 (wedge). Cells were harvested at 18 hpi and detected using immunoblot analysis with anti-MAVS, anti-NP, or anti-β-actin antibody. (C) A549 cells were mock treated or infected with NDV Herts/33 strain at an MOI of 5. Cells were harvested at 6, 12, and 18 hpi and detected using immunoblot analysis with anti-MAVS, anti-NP, or anti-β-actin antibody. (D) A549 cells were mock treated or infected with NDV Herts/33 strain at an MOI of 0.01, 0.1, 1, or 5 (wedge). Cells were harvested at 18 hpi and detected using immunoblot analysis with anti-MAVS, anti-NP, or anti-β-actin antibody. (E and F) HeLa cells were mock treated or infected with NDV LaSota (E) or ZJ1 (F) strain at an MOI of 1. Cells were harvested at 6, 12, 18, and 24 hpi and detected using immunoblot analysis with anti-MAVS, anti-NP, or anti-β-actin antibody. (G) HEK-293T was transfected with Flag-tagged chicken MAVS (cMAV) and human MAVS (hMAVS). At 24 h after transfection, cells were infected with NDV at an MOI of 1. Cells were harvested at 12 and 24 hpi and detected using immunoblot analysis with anti-Flag, anti-NP, or anti-β-actin antibody. (H) HeLa cells were mock treated or infected with NDV (MOI of 1) or UV-treated NDV (MOI of 10). Cells were harvested at 18 hpi and detected with anti-MAVS, anti-NP, or anti-β-actin antibody. (I to K) HeLa cells were mock treated or infected with NDV at an MOI of 1. Cells were harvested at 6, 12, 18, and 24 hpi and detected using qRT-PCR with MAVS (I), IFN-β (J), or IFIT1 (K) primers. (L) HeLa cells were mock infected or infected with NDV at an MOI of 1 and maintained in the presence or absence of the lysosome inhibitor CQ (50 μM) or the autophagy inhibitor wortmannin (Wort; 100 nM) for 12 h. Cells were harvested and detected using immunoblot analysis with anti-MAVS, anti-NP, anti-LC3, or anti-β-actin antibody. For proteasome inhibition assay, the infected cells were treated with MG132 (20 μM) for 6 h prior to immunoblot analysis. (M) HEK-293T cells were cotransfected with HA-MAVS and Flag-ubiquitin. At 24 h after transfection, cells were infected with NDV at an MOI of 1. At 12 and 24 hpi, cells were harvested, immunoprecipitated (IP) with anti-HA antibody, and further detected using immunoblot analysis with anti-Flag antibody. Expression levels of the proteins were analyzed by immunoblot analysis of the lysates with anti-HA or anti-β-actin antibody. (N) HEK-293T cells were cotransfected with Myc-MAVS and either HA-ubiquitin (K48) or HA-ubiquitin (K63). At 24 h after transfection, cells were infected with NDV at an MOI of 1 and maintained in the presence or absence of the proteasome inhibitor MG132 (20 μM, 6 h prior to immunoprecipitation). At 18 hpi, cells were harvested, immunoprecipitated with anti-Myc antibody, and further detected using immunoblot analysis with anti-HA antibody. Expression levels of the proteins were analyzed by immunoblot analysis of the lysates with anti-Myc or anti-β-actin antibody. Data are presented as means from three independent experiments. #, P > 0.05; ***, P < 0.001.