Fig 3.
SPOP lowers EV71-2Apro protein stability. (A) The relative mRNA level of EV71-2Apro was measured by RT-qPCR in HEK293T cells co-transfected with the vectors to express Flag-2A and empty vectors (−) or HA-SPOP. (B) The relative mRNA level of EV71-2Apro was measured by RT‒qPCR in HEK293T cells co-transfected with vectors to express Flag-2A and control shRNAs (−) or shSPOP (1# and 2#). (C) HEK293T cells were transfected with a vector to express Flag-2A. Thirty-six hours after transfection, cells were treated with CHX (100 µg/mL) for 0, 2, 4, 8, and 12 hours. The protein levels of Flag-2A were analyzed by immunoblotting. (D) HEK293T cells transfected with Flag-2A plasmids were pretreated with CHX (100 µg/mL) for 6 hours and then treated with DMSO or MG132 (10 µM) for 4 hours. The protein levels of Flag-2A were analyzed by immunoblotting. (E) HEK293T cells transfected with Flag-2A plasmids were pretreated with CHX (100 µg/mL) for 6 hours and then treated with DMSO or MA (20 mM) for 8 hours. The protein levels of Flag-2A were analyzed by immunoblotting. (F) HEK293T cells were transfected with either empty vectors (CON) or HA-SPOP plasmids, together with Flag-2A plasmids. Then, the cells were treated with CHX for 0, 6, and 12 hours. Whole-cell extracts were analyzed by immunoblotting using the indicated antibodies (left). Data were analyzed by GraphPad Prism 7 (right). N.S., not significant (P > 0.05) and ***P < 0.001 (two-tailed unpaired Student’s t-test). Data are representative of three independent experiments (C–F) or shown as the mean and s.d. of three biological replicates (A and B).