Fig 1: Cul3 and Rbx1 interact with NSP1 and contribute to ß-TrCP degradation.(A) Lysates of HEK293 cells co-transfected with GFP-NSP1, Myc-Cul3, and HA-Rbx1 were subject to immunoprecipitation using a-Myc or a-HA antibody and probed for a-GFP antibody. (B) HEK293-Wa-NSP1 stable cell lines were treated with 1 µg/ml of doxycycline for 24 hr and harvested for immunoprecipitation using a-GFP antibody and probed for indicated antibodies. (C) Lysates of mock or Wa infected HEK293 cells (MOI = 3, 24 hpi) were subject to immunoprecipitation using control rabbit polyclonal or a-Cul3 antibody, and probed for indicated antibodies. (D) HEK293 cells stably expressing RRV or ETD-NSP1 were transfected with indicated siRNA, treated with doxycycline, and harvested for western blot using indicated antibodies. (E) Same experiment as in (D) except that HEK293 cells stably expressing Wa-NSP1 were used instead (HD1: HECTD1). Blots were quantified and the level of ß-TrCP is normalized to loading control GAPDH. The ratio in mock-treated cells is set to 1. (F) Same experiment as in (D) except that ST3, UK, or SA11-5S NSP1s were used (HD1: HECTD1). (G) HEK293 cells stably expressing Wa-NSP1 were treated with MLN4924 and doxycycline, harvested for western blot using indicated antibodies. In all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p=0.05; **p=0.01; ***p=0.001).