hyde/PBS for 20 minutes. Larvae were washed and then incubated in 2N HCL for 30 minutes, 100 mM borax for 2 minutes, washed and then blocked and stained as described above. Confocal stacks of 0.5 lm intervals were analyzed using Volocity 3DM software (Improvi- sion) for cell cycle and apoptosis assays and ImageJ for the replication assay. To quantitate the BrdU stainings, the mean grayscale value of three evenly-spaced regions of 1,400 square pixels in the wide nuclei- dense bands of the brain lobe were averaged for each brain lobe. Co-immunoprecipitation and Western blots. Epitope-tagged Rad9 and Brca2 were expressed using the germline Gal4 driver nanos-VP16 [61]. 10 ovary pairs were ground in 150 ll IP buffer (50 mM Tris pH 8, 150mM NaCl, 1mM CaCl2, 0.1% Tween-20) supplemented with complete mini protease inhibitor EDTA-free (Roche) and type I and II phosphatase inhibitors (Sigma). Cleared lysate was incubated at 4 8C for 2 hours with mouse aFLAG M2 antibody (Sigma) bound to 20 lL Protein A/G PLUS Agarose slurry (Santa Cruz Biotechnology) after a 30 minute preincubation with an equal volume of Protein A/G beads without antibody. Fusion proteins were detected using mouse aFLAG-M2-HRP conjugate (Sigma) at 1:2000 and rat aHA-HRP conjugate (Roche) at 1:3000 for Western analysis. Supporting Information Figure S1. brca2 Is Not Required for the Checkpoints That Respond to Irradiation Damage in the Drosophila Larval Wing Discs and Brain Lobes Climbing third instar larvae were irradiated and then dissected prior to ﬁxation. Representative images are projections of 0.5 micron interval stacks of control tissues (A-R) or tissues that were irradiated prior to ﬁxation (A’-R’). (A,E,I,M,A’,E’,I’,M’) Larvae were ﬁxed 1 hour after a 4,000 Rad IR exposure and stained for the mitotic marker P- H3. 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