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. 2020 Feb 6;16(2):e1008293. doi: 10.1371/journal.ppat.1008293

Fig 6. USP27X deubiquitinates the K63-linked ubiquitination of RIG-I.

Fig 6

(A–C) HEK293T cells were transfected with USP27X and RIG-I(N) together with HA-tagged wild-type Ub (HA-Ub) (A), HA-Ub-K63 (B), or HA-Ub-K48 (C) plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting analysis with the indicated antibodies. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels. (D) Purified Flag-USP27X and K63-linked ubiquitinated Flag-RIG-I(N) were incubated together in deubiquitination buffer, followed by immunoblotting. (E) HEK293T cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cells were infected with SeV for 9 h. Cell lysates were immunoprecipitated with anti-Flag beads and immunoblotted with the indicated antibodies. (F) HEK293T USP27X+/+ and USP27X-/- cells were infected with SeV for 9 or 12 h, lysed, and immunoprecipitated with anti-RIG-I antibody followed by immunoblotting. (G) HEK293T cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting.