FIG 9.

The ANDV-NSs protein reduced MAVS polyubiquitination in cells. HEK 293T cells were cotransfected with HA-EV or HA-NSs plus Flag-MAVS and HA-ubiquitin vectors. At 24 h posttransfection, cells were treated with MG-132 (5 μM) for 6 h and lysed. (A) Whole-cell lysate (WCL) was used to evaluate total ubiquitinated proteins as well as the overexpression of MAVS and the ANDV-NSs proteins. GAPDH was detected as a loading control. (B) MAVS IP fractions were generated from the WCL by immunoprecipitation assay using an anti-MAVS antibody. Western blotting was performed using a mouse anti-HA to visualize ubiquitinated MAVS and an anti-MAVS to confirm MAVS immunoprecipitation. Horseradish peroxidase (HRP)-conjugated protein A/G was used to detect the primary antibodies.