FIG 5.

The presence or absence of USP18 regulates the level of p21. (A) PMA-differentiated USP18KO THP-1 cells and vector control cells were stimulated with 1,000 U/ml of IFN-β. At 24 h after stimulation, the cells were lysed and immunoblotted for the endogenous expression of p21, total and phosphorylated SAMHD1, USP18, and GAPDH as a loading control. (B) After 48 h of PMA treatment, the cells were also tested for HIV-1 replication and transduced with HIV-1 reporter virus in the presence or absence of copackaged VPX. (C) In a related experiment, PMA-differentiated cells USP18KO and control cells were treated with PMA and subsequently immunoblotted for p21, total, and phosphorylated SAMHD1, SKP2, USP18, and GAPDH as a loading control. (D) PMA-differentiated SAMHD1KO THP-1 cells expressing the vector control and USP18 were immunoblotted for p21, USP18, SAMHD1, and GAPDH as a loading control. These cells were treated with 25 ng/ml PMA. (E) At 48 h posttreatment, the cells were transduced with a single round of HIV-1 reporter virus, and the luciferase activity was measured after 72 h. (F) In a related experiment, the cells were tested for SKP2 protein expression levels by immunoblotting the PMA-differentiated vector control and USP18 expressing SAMHD1KO THP-1 cells for p21, SAMHD1, SKP2, USP18, and GAPDH as a loading control. Each panel is representative of at least two independent experiments.