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

(A) PAN-, K57- or vIL-6-reporter was co-expressed with HA-K-RTA and FLAG-OTUD4 WT or C45A mutant in HEK293T cells. Luciferase activities were determined at 24 h post-transfection. (B) iSLK cells transduced with sh-Ctrl (Scramble) or sh-OTUD4 were treated with DMSO, Baf-A1 (1 mM) or MG132 (10 μM), followed by Dox (0.2 μg/ml) induction for 12 h. WCL were collected and analyzed by immunoblotting. (C) HEK293T cells transduced with sgRNA targeting ATG7 were co-transfected with K-RTA and FLAG-OTUD4/C45A (0, 0.5, 1 or 2 μg), followed by immunoblotting at 24 h post-transfection. (D) HEK293T cells were co-transfected with FLAG-K-RTA, HA-Ub (K63-only) and Myc-OTUD4/C45A, and then treated with MG132 (10 μM). Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (E) HEK293T cells were co-transfected with FLAG-Myd88, HA-Ub (K63-only) and Myc-OTUD4/C45A. Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (F) HEK293T cells were co-transfected with HA-OTUD4 and FLAG-K-RTA or the indicated mutants, followed by immunoblotting at 24 h post-transfection. (G) FLAG-K-RTA WT or the indicated mutants were co-expressed with HA-Ub (K48-only) in HEK293T cells. Then denatured immunoprecipitation was performed as described in S3D Fig.

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