Fig 2.
SPOP is a negative regulator of EV71-2Apro. (A) HEK293T cells were co-transfected with vectors to express Flag-2A and increased amounts (0.5 and 1.0 µg) of HA-SPOP. Whole cell extracts were subjected to immunoblotting as indicated. (B) HEK293T cells were co-transfected with vectors to express Flag-2A and control shRNAs (−) or two shRNAs against SPOP (shSPOP, 1# and 2#). Seventy-two hours after transfection, whole cell extracts were analyzed by immunoblotting using the indicated antibodies (left). The relative mRNA levels of SPOP were measured by RT-qPCR in HEK293T cells transfected with control shRNA (−) or shSPOP (1# and 2#) (right). (C) HEK293T cells were transfected with vectors to express Flag-2A and either HA-SPOP wild type (WT) or HA-SPOP-F102C mutant plasmids. Flag-2A protein levels were analyzed by immunoblotting as indicated. (D) HEK293T cells were transfected with either Flag-2B or Flag-2C plasmids, together with empty vectors (−) or increasing amounts of HA-SPOP plasmids. Whole cell extracts were analyzed by immunoblotting using the indicated antibodies. Data are representative of three independent experiments with similar results (A–D).