OTUD4 bridges the association of K-RTA and USP7 to promote KSHV lytic reactivation.

(A) WCLs were collected from HEK293T cells transfected with the indicated plasmids and subsequently subjected to immunoprecipitation with anti-FLAG affinity agarose, followed by immunoblotting. (B) Immunoblotting analysis of HEK293T cells transduced with sh-Ctrl or sh-USP7. (C) HEK293T-shUSP7 cells as described in S6B Fig. were co-transfected with FLAG-K-RTA, HA-Ub (K48-only) and Myc-OTUD4/C45A, and then treated with MG132 (10 μM). Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (D) HEK293T cells were co-transfected with FLAG-OTUD4 full length (FL) or 1-425aa with HA-K-RTA, followed by immunoblotting at 24 h post-transfection. (E) HEK293T cells were co-transfected with FLAG-K-RTA or FLAG-K-RTA-K218R, HA-Ub (K48-only), and Myc-OTUD4 full length or 1-425aa, and then treated with MG132 (10 μM). Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (F) SLK.iBAC-GFP stable cells as described in Fig 6I were induced with Dox (1 μg/ml) for 48 h, and WCLs were analyzed by immunoblotting. (G) FLAG-OTUD4 and HA-OTUD4 or FLAG-OTUD4 (1-425aa) and HA-OTUD4 (1-425aa) were co-expressed in HEK293T cells, and co-immunoprecipitation and immunoblotting were performed at 24 h post-transfection.

(TIF)