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. 2020 Nov 25;48(22):12711–12726. doi: 10.1093/nar/gkaa1090

Figure 4.

Figure 4.

USP36 deubiquitinates PrimPol. (A) HEK293T cells stably expressing control (Ctrl) or USP36 shRNAs were transiently transfected with Myc-tagged PrimPol. After 48 h, cells were treated with MG132 (50 μM) for 3 h. Cell lysates were subjected to immunoprecipitation with anti-Myc agarose beads, and then blotted with the indicated antibodies. (B) HEK293T cells stably expressing Ctrl or USP36 shRNAs were transiently transfected with HA-tagged ubiquitin. After 48 h, cells were treated with MG132 (50 μM) for 3 h. Cell lysates were subjected to immunoprecipitation with anti-HA agarose beads, and then blotted with the indicated antibodies. (C) HEK293T cells with USP36 stably knocked-down were transiently transfected with indicated constructs. After 48 h, cells were treated with MG132 (50 μM) for 3 h before collecting. Cell lysates were subjected to immunoprecipitation with anti-HA agarose beads, and then blotted with the indicated antibodies. (D) Deubiquitination of PrimPol in vitro by USP36. Ubiquitinated PrimPol was incubated with purified USP361–800-WT or USP361–800-CA in vitro. Cell lysates were subjected to immunoprecipitation with anti-Myc agarose beads, and then blotted with the indicated antibodies.