Fig 1.
EV71-2Apro interacts with the host ubiquitin E3 ligase SPOP. (A) Alignment of a putative SPOP-binding motif in EV71-2Apro. (B and C) Immunoprecipitation analysis of the interaction between SPOP and 2Apro in HEK293T cells co-transfected with HA-SPOP and Flag-2A plasmids. WCL, whole cell lysate. (D) HEK293T cells were transfected with a vector to express Flag-2A. Immunoprecipitation was performed using the Flag affinity gel, and then endogenous SPOP was detected by immunoblotting with an anti-SPOP antibody. (E) HEK293T cells were transfected with a vector to express Flag-2A. Immunoprecipitation was performed using the SPOP antibody, and Flag-2A was detected by immunoblotting with an anti-Flag antibody. (F) The domains of the SPOP protein and SPOP deletion mutants. (G, H, and I) Immunoprecipitation analysis of the interaction between SPOP mutants and 2Apro in HEK293T cells co-transfected with Flag-2A and either HA-SPOP wild type (WT), HA-SPOP-ΔMATH mutant, HA-SPOP-ΔBTB mutant or HA-SPOP-F102C mutant plasmids. (J) Immunoprecipitation analysis of the interaction between SPOP and 2Apro in HEK293T cells co-transfected with HA-SPOP and Flag-2A (WT or the ΔVSSTT mutant). (K) Immunofluorescence analysis of the co-localization of HA-SPOP and Flag-GFP-2A in HeLa cells. Data are representative of three independent experiments with similar results (B–J).