KSHV K-RTA interacts with OTUD4.

(A) HEK293T cells were transfected with the indicated plasmids, and whole cell lysates (WCLs) were collected for immunoprecipitation with anti-FLAG affinity agarose. The input and precipitated samples were analyzed by immunoblotting. (B) SLK.iBAC-GFP cells were induced with Doxycycline (Dox, 1 μg/ml) for 24 h to trigger lytic reactivation, and co-immunoprecipitation and immunoblotting were performed with the indicated antibodies. (C) SLK.iBAC cells stably expressing vector control or FLAG-OTUD4 were induced with Dox (1 μg/ml) for 24 h, and immunofluorescence was performed using anti-FLAG and K-RTA antibodies. Scale bars, 20 μm. (D-E) HEK293T cells were transfected with the indicated plasmids, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose, followed by immunoblotting. (F) The interaction between K-RTA truncations, including K-RTA(1–210), K-RTA(1–489), K-RTA(211–691), and K-RTA(490–691), and OTUD4 was assessed by co-immunoprecipitation in HEK293T cells. (G) The interaction between K-RTA point mutations, including K-RTA(G677A/T678A/L679A), K-RTA(Y680A/Q681A/L682A), K-RTA(H683A/Q684A/W685A), K-RTA(R686A/N687A/Y688A), and K-RTA(F689A/R690A/D691A), and OTUD4 was assessed by co-immunoprecipitation in HEK293T cells.