86 | Chapter 4 mM b-glycerophosphate, and 100µM phenylmethanesulfonylfluoride (PMSF). The protein pellets were homogenized passing through a 27G needle and protein concentrations were measured using the bicinchoninic acid (BCA) assay (Sigma-Aldrich). For each sample, 15 mg of protein lysate was used for protein separation in PreciseTM 4-20% gradient Tris-SDS gels (Thermo Fisher Scientific, Landsmeer, The Netherlands). Subsequently, separated protein lysates were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, EMD Millipore), membranes were blocked in 5% BSA (BSA Fraction V, Roche Life Sciences, Almere, the Netherlands) in TBS-T or 5% milk in TBS-T for 2 hours. Primary antibodies were incubated overnight at 4ºC. Following a series of thorough washes with TBS-T, membranes were incubated with secondary antibodies (goat-anti-rabbit-HRP or rabbit-anti-mouse HRP, DAKO, Agilent Technologies, Heverlee, Belgium) for 2 hours at room temperature. Antibody binding (protein expression) was visualized using Amersham ECL Prime Western Blotting detection reagent (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The following antibodies were used in this study: Rabbit-anti-RAG1 (D36B3), rabbit-anti-FOXO1 (C29H4), rabbit-anti-phospho-FOXO1 (Ser256) (9461), rabbit-anti-AKT (4691), rabbit-anti-phospho-AKT (Ser473) (D9E), rabbit-anti-phospho-p53 (Ser15) (9284), rabbit-anti-mTOR (2972), rabbit-anti-phospho-mTOR (Ser2448) (D9C2), rabbit-anti-cleaved caspase-3 (Asp175) (9661), rabbit-anti-GADD45a (D17E8) (all from Cell Signaling Technology, Danvers, MA), mouse-anti-phospho-ATM (Ser1981) (10H11.E12), and mouse-anti-b-actin (C4) (both from EMD Millipore). For immunoprecipitation, nuclear extracts from 60x106 cells were prepared according to the nuclear complex co-IP kit (active Motif, Carlsbad, CA). Cleared lysates were incubated overnight at 4°C with rabbit anti-phospho-(Ser/Thr) ATM/ATR substrate antibody (2851, Cell Signaling Technology), or polyclonal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX) with 1% detergent in 150 mM NaCl. Antibody-bound proteins were captured by protein-A sepharose beads (CL-4B, GE Healthcare Bio-Sciences AB), thoroughly washed, resolved in Novex Bolt™ 10% Bis-Tris Plus gels (Thermo Fisher Scientific), and subjected to Western blotting. The following antibodies were used: rabbit-anti-FOXO1 (C29H4, Cell Signaling Technology), and rabbit-anti-NBS1 (Y112, Abcam, Bristol, UK). Real-time quantitative RT-PCR Levels of mRNA expression were determined using the CFX384 (Bio-Rad, Hercules, CA) real-time PCR platform. Briefly, RNA from cells was isolated by TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol. For each sample cDNA was synthesized from 500 ng RNA, using random primers (Promega Benelux, Leiden, the Netherlands) and M-MLV reverse transcriptase (Invitrogen-Life Technologies, Bleiswijk, the Netherlands). For each sample, cDNA was diluted 1:5 with PCR-grade water. The PCR mastermix contained 5 ml of Sso Fast EvaGreen supermix (Bio-Rad, Hercules, CA), and 0.5ml of 10 mM reverse and forward primers (Biolegio, Nijmegen, the Netherlands). The final volume was adjusted to
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