Anti mre11

BMDM and MEFs were lysed in RIPA buffer and whole cell lysates were generated with LDS sample buffer (Invitrogen) supplemented with dithiothreitol (DTT). For analysis of culture supernatants, protein was precipitated with 7.2% w/v trichloroacetic acid (TCA) (Sigma) followed by two acetone washes. Immunoblotting was carried out as previously described (Helmink et al., 2011 (link)). Primary antibodies used were anti-γ-H2AX clone JBW301 (Millipore) (RRID:AB_309864), anti-H2AX (Millipore) (RRID:AB_2233033), anti-phospho-KAP-1 (Bethyl Laboratories) (RRID:AB_669740), anti-KAP-1 (GeneTex) (RRID:AB_372041), anti-caspase 1 (p20, Casper-1) (Adipogen) (RRID:AB_2490248), anti-DNA-PKcs (Invitrogen), anti-Ku70 (Cell Signaling Technology), anti-Ku80 (Cell Signaling Technology) (RRID:AB_2257526), anti-ATM clone MAT3 (Sigma), anti-Mre11 (Novus) (RRID:AB_10077796), anti-Nbs1 (Abcam) (RRID:AB_777006), anti-Rad50 (Abcam) (RRID:AB_2176935), anti-ATR (Novus) (RRID:AB_10003234), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma). Secondary reagents were horseradish peroxidase–conjugated anti–mouse IgG (Promega) or horseradish peroxidase–conjugated anti- rabbit IgG (Cell Signaling Technology).

Ten million bulk testicular cells were fixed in 1% formaldehyde (Sigma, F1635) at 37 °C for 10 min. Fixation was quenched with glycine (Sigma) at a final concentration of 125 mm. Cells were washed twice with cold PBS, and pellets were snap frozen on dry ice and stored at −80 °C until sonication. Sonication, immunoprecipation, and library preparation were performed as previously described in (Canela et al.^{16 (link)}). In brief, frozen pellets were resuspended in 1 mL cold RIPA buffer (10 mm Tris-HCl pH 7.5, 1 mm ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulphate, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 Complete Mini EDTA-free proteinase inhibitor tablet (Roche)) and sonicated using a Covaris S220 at duty cycle 20%, peak incident power 175, and cycle/burst 200 for 30 min at 4 °C.
Chromatin was precleared with 40 μL prewashed Dynabeads Protein A (ThermoFisher) for 30 min at 4 °C, followed by incubation with 40 μL Dynabeads Protein A bound to either 6 μL anti-H3K4me3 (Millipore, 07–473) or 4 μL anti-MRE11 (Novus, NB100-142) overnight at 4 °C. Beads were washed and cross-linking reversed the next day as described in (Canela et al.^{16 (link)}). Immunoprecipitated DNA was removed from beads and stored at −20 °C until library preparation, which was performed as described in (Canela et al.^{16 (link)}).

Cells were lysed in RIPA Lysis buffer (Millipore) supplemented with 0.1% SDS, protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktails (Sigma). Samples were prepared using NuPage LDS Sample buffer (Invitrogen) with 100 mM DTT, and proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using the NuPage MOPS system (Invitrogen). Antibodies used were as follows: anti-Mre11 (Novus Biological, NB 100–142, 1:2000), anti-NBS1 (p95/NBS1, Cell Signaling #3002, 1:1000), anti-Rad50 (Novus Biological, NB 100–154, 1:500), anti-pATM S1981(Cell Signaling #4526, 1:500), anti-pATR S1989 (Kerafast, 1:500), anti-Chk1 S317 (Cell Signaling, #2344, 1:400), anti-Chk1 S345 (Cell Signaling, #2348, 1:400).