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. 2009 Jul 31;5(7):e1000530. doi: 10.1371/journal.ppat.1000530

Figure 1. BZLF1 protein associates with Cul2 and Cul5 through the Cul-box motif.

Figure 1

(A) BZLF1 protein contains putative Cul2-box and Cul5-box motifs at the N-terminus. Conserved amino acids are indicated in bold. TA, transactivation domain; bZip, basic leucine-zipper domain. Mutated amino acids are highlighted. (B and C) Lysates of 293T cells transiently expressing the indicated proteins were subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies. (D) BZLF1 protein interacts with both Cul2 and Cul5 in lytic replication-induced cells. The lytic infection was induced by transfection with BZLF1 protein expression plasmid into 293/EBV cells. Cells were treated with MG132 for 4 h before harvesting and then harvested 48 h post-transfection. IP and IB assays were performed using the indicated antibodies. (E) The BZLF1 M3 mutant features relaxed reduction of p53. SaOS-2 cells were transfected with BZLF1 wild-type or its mutant expression plasmid, and p53 expression plasmids as indicated. Lysates were applied for immunoblot analysis with each antibody indicated. The intensity of the bands is expressed as a ratio between p53 and GAPDH. (F) Cul2 and Cul5 are required for p53 degradation. 293/EBV cells were co-transfected with plasmids expressing BZLF1 protein and shRNA targeting Cul2 and Cul5 mRNAs. Cells were lysed 48 h post-transfection for IB analysis with the indicated antibodies.