Figure 4.

RFWD3 promotes ZRANB3 recruitment to nascent DNA at stalled replication forks. (A) Detection of RFWD3 and HA-ZRANB3 levels in U2OS cells used in Fig. 4, B and C. Immunoblot was performed with antibodies against RFWD3 and HA. (B) U2OS cells expressing HA-ZRANB3 were transfected with the indicated siRNAs, pulsed with 10 µM EdU for 10 min, treated with 1 µg/ml 4NQO for 4 h, and fixed. Biotin was conjugated to EdU by click chemistry, and proximity ligation assay (PLA) was performed with anti-HA and anti-biotin antibodies. Images are representative of results quantitated in Fig. 4 C. Scale bars, 5 µm. (C) Box plot depicting PLA foci per cell for each condition (>200 cells) in Fig. 4 B. Whiskers represent the 10th and 90th percentiles (****P < 0.0001, Mann Whitney test). (D) Detection of RFWD3 and HA-ZRANB3 levels in U2OS cells used in Fig. 4, E and F, and Fig. S2 F. Immunoblot was performed with antibodies against RFWD3 and HA. (E) U2OS cells expressing HA-ZRANB3 were transduced with shRFWD3-4 or non-targeting shFF along with either shRNA-resistant WT or C315A RFWD3. They were then pulsed with 10 µM EdU for 10 min, treated with 1 µg/ml 4NQO for 4 h, and fixed. Biotin was conjugated to EdU, and PLA was performed with antibodies against HA and biotin. Scale bars, 5 µm. (F) Box plot of PLA foci per cell for each condition (>200 cells) in Fig. 4 E. Whiskers represent the 10th and 90th percentiles (n.s., not significant; ****P < 0.0001; Mann Whitney test).