Figure 2.

USP36 is a PrimPol binding protein. (A) Interaction between endogenous USP36 and PrimPol. HEK293T cell were collected and subjected to immunoprecipitation using control IgG and anti-USP36 antibodies. Blots were then probed with the indicated antibodies. (B) Interaction between exogenous USP36 and endogenous PrimPol. Cell lysates from HEK293T cells transiently transfected with Flag-USP36 were subjected to immunoprecipitation with anti-Flag agarose beads, and then blotted with the indicated antibodies. (C) HEK293T cells were transiently transfected with Flag-USP36 for 48h, 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 were blotted with the indicated antibodies. (D) Quantification of the amount of PrimPol in the pull-down relative to the PrimPol input level. The graphs represent mean ± SD, two-tailed, Student's t-test. *P < 0.05. (E) In situ PLA between endogenous USP36 and PrimPol after treated with/without HU (10 mM) for 4 h in OVCAR8 cells. Representative images are shown with merged PLA and nuclei (DAPI) channels from PLA experiments. Scale bar in the bottom left is 10 μm. **P < 0.01. Each red dot represents the detection of USP36-PrimPol interaction complex, and the mean ± SD are shown in (F). (G) The N-terminal USP domain of USP36 is required for binding to PrimPol. HEK293T cells were transiently transfected with Myc-PrimPol together with Wild-type (WT) and truncated mutants (1-420, 1–800, 421–800 and 801–1121aa) of Flag-USP36. The protein interaction was assayed by immunoprecipitation with anti-Flag agarose beads, and then blotted with the indicated antibodies. Schematic representation of the Flag-USP36 and its deletion mutants are shown in (H). USP: Ub-specific protease domain. CTD: Carboxy-terminal domain.