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

Figure 3.

Figure 3.

USP36 interacts with PrimPol is dependent on its ubiquitination sites K329 and K338 upon DNA replication stress. (A) USP36 stably knocked-down HEK293T cells were transiently transfected with Flag-USP36 and HA-Ub for 48h. Cells were pretreated with HU (10 mM) for 5h, then cotreated with MG-132 (50 μM) for additional 3 h. Cell lysates were subjected to immunoprecipitation with anti-Flag agarose beads, and then blotted with the indicated antibodies. (B) USP36 stably knocked-down HEK293T cells were transiently transfected with Flag-USP36 and HA-tagged Ub-K48 for 48h. Cells were pretreated with HU (10 mM) for 5h, then cotreated with MG132 (50 μM) for additional 3 h. Cell lysates were subjected to immunoprecipitation with anti-Flag agarose beads, and then blotted with the indicated antibodies. (C) Identification of the ubiquitination sites of USP36 for its K48-specific polyubiquitination. USP36 stably knock-down HEK293T cells were transiently transfected with indicated constructs. After 48 h, cells were treated with MG-132 (50 μM) for 3 h before collecting. Cell lysates were subjected to immunoprecipitation with anti-Flag agarose beads, and then blotted with the indicated antibodies. (D) Flag-tagged USP36 expression constructs and Myc-PrimPol plasmid were transfected into USP36 stably knock-down HEK293T cells. After 48 h, cells were treated with MG-132 (50 μM) for 3 h before collecting. Cell lysates were then immunoprecipitated with anti-Flag beads and immunoblotted as indicated. (E) USP36 stably knocked-down HEK293T cells were transiently transfected with indicated constructs, then left untreated or treated with HU (10 mM) for 8 h. The cell lysates were subjected to immunoprecipitation with anti-Flag agarose beads, and immunoprecipitated lysates were equalized for Flag-USP36. Then cell lysates blotted with the indicated antibodies. (F) Quantification of the amount of Myc-PrimPol in the pull-down relative to the Myc-PrimPol input level. The graphs represent mean ± SD, two-tailed, Student's t-test. *P < 0.05; NS = not significant. (G) Representative images of merged PLA and nuclei (DAPI) channels from PLA experiments. USP36 stably knock-down OVCAR8 cells were transiently transfected with USP36-wild-type (WT) or the USP36-WT 2KR mutant, then left untreated or treated with HU (10 mM) for 4 h. In situ PLA was used to assess the interaction between USP36 and endogenous PrimPol. Scale bar in the bottom left is 10 μm. Each red dot represents the detection of USP36-PrimPol interaction complex, and the graphs represent mean ± SD are shown in (H). **P < 0.01; NS = not significant.