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. 2019 Aug 28;5(8):eaax1031. doi: 10.1126/sciadv.aax1031

Fig. 5. Viperin-induced methionine oxidation stabilizes MCM7 and promotes DNA replication.

Fig. 5

(A) Reciprocal co-IP assays to examine physical interactions between viperin and MCM7. (B) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (C) 293T cells were transfected with plasmids containing indicated genes. At 24 hours after transfection, cells were treated with CHX (10 μg/ml). WCLs were then analyzed by immunoblotting. (D) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (E) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, cells were harvested, cross-linked, and used for ChIP assay to determine relative binding of MCM7 wild type and MCM7-TA at cellular c-Myc replication origin. (F) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, cells were labeled with EdU (5-ethynyl-2′-deoxyuridine), total DNA was extracted, and Click-iT procedure was used to isolate DNA from the active sites of replication. DNA was then analyzed by qPCR at cellular c-Myc replication origin. (G) 293T cells were transfected with plasmids containing indicated genes after transfection for 6 hours with siRNA, as indicated. At 48 hours after transfection, cells were harvested, cross-linked, and used for ChIP assay to determine relative binding of MCM7 wild type and MCM7-TA at cellular c-Myc replication origin. (H) 293T cells were transfected with plasmids containing indicated genes after transfection for 6 hours with siRNA, as indicated. At 48 hours after transfection, cells were labeled with EdU, total DNA was extracted, and Click-iT procedure was used to isolate DNA from the active sites of replication. DNA was then analyzed by qPCR at cellular c-Myc replication origin. (I) In vivo co-IP assays to detect the interaction between MCM7 and viperin. The WCLs of 293T were precipitated with anti-viperin. Precipitated proteins and WCLs were analyzed by immunoblotting. (J) 293T cells were transfected with siRNA, as indicated. At 48 hours after transfection, WCLs were then analyzed by immunoblotting. (K) 293T cells were transfected with siRNA, as indicated. At 48 hours after transfection, cells were harvested, cross-linked, and used for ChIP assay to determine relative binding of endogenous MCM7 at cellular c-Myc replication origin. (L) 293T cells were transfected with siRNA as indicated. At 48 hours after transfection, cells were labeled with EdU, total DNA was extracted, and Click-iT procedure was used to isolate DNA from the active sites of replication. DNA was then analyzed by qPCR at cellular c-Myc replication origin. For (E) to (H), (K), and (L), the data are expressed as means ± SEM; n = 3; **P < 0.01, ***P < 0.001, and ****P < 0.0001.