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. 2022 Jan 20;298(3):101611. doi: 10.1016/j.jbc.2022.101611

Figure 3.

Figure 3

TRIM34 promotes IAV induced K63-linked polyubiquitination of ZBP1.A, 293T cells were transfected with Flag-TRIM34 or/and Myc-ZBP1 and the indicated ubiquitin plasmids for 48 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. B, THP-1 macrophages were transfected with plasmids or siRNAs for 24 h. Then, cells were infected with IAV (MOI = 1) for 24 h or left uninfected. Co-IP and immunoblot analysis were performed with the indicated antibodies. C and D, experiments were performed similar to those in (B), except HA-Ub (K63) plasmid (C) or HA-Ub (K48) plasmid (D) were used. E, 293T cells were transfected with the indicated plasmids for 48 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. F, ZBP1, ZBP1 (K17A), ZBP1 (K43A), and TRIM34 were translated in vitro, and biotin-ubiquitin, E1, and the indicated E2s were added for ubiquitination assays. Ubiquitin-conjugated proteins were detected by immunoblot with horseradish peroxidase-streptavidin. Before ubiquitination analysis, the input levels of the translated proteins were detected by immunoblots. All experiments were repeated at least three times. IAV, influenza A virus; TRIM, tripartite motif; ZBP1, Z-DNA-binding protein 1.