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. 2014 Jun 2;211(6):1027–1036. doi: 10.1084/jem.20131939

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Figure 3. — CDK-dependent phosphorylation of CtIP^T847 is essential for CtIP function. (A) WT and BRCA1^Δ11/Δ11 MEFs were irradiated with a 364-nm laser line derived from a LSM510 microscope. After 10-min recovery, cells were processed for immunofluorescence analysis of CtIP (red) and γH2AX (green). Bar, 15 µm. (B) Western blot showing expression of transgenic CtIP^WT and CtIP^S327A protein and phosphorylated KAP1 and p53 in B cells. (C) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation. Three independent experiments are reported. For each experiment, >600 cells were counted (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (D) Analysis of total genomic instability in metaphases from B cells isolated from mice of indicated genotypes after treatment for 16 h with 1 µM PARPi. The mean number of chromosome aberrations per cell measured in 3 independent experiments is shown (n = 250 metaphases; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (E) Levels of transgenic CtIP^WT, CtIP^T847E, and CtIP^T847A protein expression and KAP1 and p53 phosphorylation in mutant B cells detected by Western blotting. (F) Analysis of total genomic instability in metaphases from B cells with or without PARPi treatment. Mean of three independent experiments is shown (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Percentage of B cells with >5 RAD51 foci after 10 Gy IR. Three independent experiments are reported. >600 cells were counted for each genotype (P = 0.0251; two-tailed paired t test). (H) Western blot showing the level of CtIP expression in infected cells using anti-FLAG antibody (top). WT MEFs expressing FLAG-CtIP^WT, FLAG-CtIP^T847A, or FLAG-CtIP^T847E were treated with Hoechst 33342 and irradiated with the 364-nm laser line and after 20-min recovery FLAG (red) and 53BP1 (green) were detected by immunofluorescence (bottom). Bar, 15 µm. (I) Western blot showing the levels of CtIP protein in B cells from mice of the indicated genotype (top). Genomic instability in B cells isolated from mice of the indicated genotypes, relative to total instability measured in BRCA1-deficient B cells. Cells were treated with 1 µM PARPi for 16 h (bottom). Three independent experiments are reported (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). For each independent experiment, one mouse per genotype was used.