OTUD4 facilitates K-RTA deubiquitination and stability by promoting K-RTA K218 deubiquitination.

(A) HEK293T cells were co-transfected with HA-K-RTA and different amounts of FLAG-OTUD4/C45A (0, 0.5, 1 or 2 μg). WCLs were collected at 24 h post-transfection and analyzed by immunoblotting. Densiometric analysis of the bands was performed with Image J. (B) HEK293T cells were co-transfected with a luciferase reporter plasmid driven by KSHV PAN promoter (PAN-Luc), HA-K-RTA and different amounts of FLAG-OTUD4 WT/C45A mutant (0, 0.1, 0.2 or 0.5 μg). Luciferase activities were determined at 24 h post-transfection. (C) HEK293T cells were co-transfected with FLAG-K-RTA, HA-Ub and Myc-OTUD4/C45A, and then treated with MG132 (10 μM). Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (D) HA-Ub (K48-only), instead of HA-Ub, was used for the detection of K-RTA ubiquitination as describe in Fig 3C. (E) HEK293T cells were co-transfected with PAN-Luc reporter, vector control or FLAG-OTUD4, and WT K-RTA or the indicated mutants. Luciferase activities were quantified at 24 h post-transfection.