Fig 1: SPOP regulates 53BP1 interaction with the p97-NPL4 complex and 53BP1 retention at DSB sites.(A and B) PC-3 (A) and 293T (B) cells transfected or not with Myc-SPOP were treated with 1 μM CPT for 3 hours and harvested for co-IP and WB. (C) Co-IP and WB of 293T cells transfected with indicated plasmids and treated with 1 μM CPT for 3 hours. (D to G) sgControl or sgNPL4 stable PC-3 cells were infected with lentivirus expressing EV, SPOP WT, or mutant F133V followed by WB (D) and IFC (E). The average 53BP1 foci number (F) and foci density (G) in each cell were quantified. Scale bar, 10 μM. Data are presented as means ± SEM of more than 300 cells from three biological replicates. *P < 0.05 and ***P < 0.001. (H to K) L3MBTL1 was depleted by a pool of siRNAs in sgControl or sgNPL4 stable PC-3 cells infected with lentivirus expressing EV and SPOP WT or mutant F133V. Cells were subjected to WB (H) and IFC (I). The average 53BP1 foci number (J) and foci density (K) in each cell were quantified, respectively. Scale bar, 10 μM. Data are presented same as aforementioned. (L and M) Lysate of PC-3 cells treated with IR (4 Gy for 1 hour) was immunodepleted with preimmune sera or antibodies for L3MBTL1 (L) or 53BP1 (M) five times followed by WB.