Figure 5
From: Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication
NS1-mediated inhibition of Rac1 SUMOylation results in reduced Rac1 activity.
(A) NS1 inhibited Rac1 SUMOylation. 293T cells were co-transfected with pcDNA3.0 or pcDNA3.0-flag-NS1 with pcDNA4.0-myc-Rac1, pXJ40-HA-SUMO-1 and UBC-9 as indicated. Anti-Rac1 antibodies were used to precipitate the cell lysates, and western blotting was conducted using HA-specific antibodies. (B) NS1 decreases SUMOylated GTP-bound Rac1. 293T cells were co-transfected with pcDNA3.0 or pcDNA3.0- flag-NS1 with pcDNA4.0- myc-Rac1, pXJ40-HA-SUMO-1 and UBC-9 as indicated. After 30 hours, the whole cell lysates were subjected to the GST-PAK pull down assay followed by western blotting using a Rac1-specific antibody. (C) Rac1 SUMOylation. 293T cells were co-transfected with pcDNA4.0-myc-Rac1, pXJ40-HA-SUMO-1 and UBC-9 as indicated. Anti-Rac1 antibodies were used to precipitate the cell lysates, and western blotting was conducted using HA-specific antibodies. (D) Rac1 SUMOylation can increase Rac1 activity. 293T cells were co-transfected with pcDNA4.0- myc-Rac1, pXJ40-HA-SUMO-1 and UBC-9 as indicated. After 30 hours, the whole cell lysates were subjected to the GST-PAK pull down assay followed by western blotting using a Rac1-specific antibody. (E) The SUMOylation sites in the Rac1 protein. 293T cells were co-transfected with pcDNA3.0, pXJ40-HA-SUMO-1, UBC-9, and the mutated Rac1 plasmids as indicated. Rac1 antibodies were used for precipitation of the lysates, and western blotting was performed with HA-specific antibodies. (F) Competition for SUMOylation by the NS1 protein inhibited Rac1 SUMOylation. 293T cells were co-transfected with pcDNA3.0 or pcDNA3.0-flag-NS1 with pcDNA4.0- myc-Rac1, pXJ40-HA-SUMO-1 and UBC-9 as indicated. Anti-Rac1 antibodies were used to precipitate the cell lysates, and western blotting was performed using HA-specific antibodies. (G) The wild type virus can inhibit Rac1 SUMOylation. 293T cells were co-transfected with pcDNA3.0 or pcDNA3.0- flag-NS1 with pcDNA4.0- myc-Rac1, pXJ40-HA-SUMO-1 and UBC-9 as indicated. Then, the cells were infected with the A/WSN/33 H1N1 virus and the mutated NS1− H1N1 virus (MOI = 0.5) for another 24 hours. Anti-Rac1 antibodies were used to precipitate the cell lysates, and western blotting was conducted using HA-specific antibodies.
