88 | Chapter 4 were exposed overnight to the concentrated virus in the presence of 7 mg/mL polybrene (Sigma-Aldrich). Transduced cells were FACS-sorted based on green fluorescent protein (GFP) expression 5 days after transduction. To assess RAG recombination activity, A70 WT mouse v-Abl pre-B cells were transduced with a retroviral RAG-reporter construct26, as described previously27. Chromatin immunoprecipitation Protein-DNA binding was studied using the MAGnifyTM Chromatin Immunoprecipitation system (Life Technologies) in accordance with the manufacturer’s protocol. Briefly, 20x106 cells were used for each IP, which were performed in triplicate for each condition. The cells were fixed in 1% formaldehyde for 5 minutes at 37ºC and quenched for 10 minutes with 125 mM glycine. Subsequently, cells were washed twice in ice-cold PBS and lysed using the enclosed lysis buffer and sonicated by Covaris S2 (intensity: 3, duty cycle: 5 %, and cycles per burst: 200) to obtain DNA fragments of an average length of 500bp. Protein concentration was measured after sonication by the BCA assay and equal amounts of fragmented chromatin-protein complexes were incubated for 2 hours with enclosed Dynabeads® that were first coupled to a cocktail of FOXO1-specific antibodies (rabbit-anti-FOXO1, C29H4 [Cell Signaling Technology], rabbit-anti-FOXO1, H-128, [Santa Cruz Biotechnology], rabbit-anti-acetyl-FOXO1, D-19, [Santa Cruz Biotechnology], rabbit-anti-FOXO1, ab70382, [Abcam], rabbit-anti-phospho-FOXO1 (Ser256), 9461, [Cell Signaling Technology]) (10 mg in total per ChIP), phospho-ATM (Ser1981) (10H11.E12, EMD MilliPore), or to polyclonal rabbit or mouse IgG (control ChIP) (Santa Cruz Biotechnology). Following a series of washes, chromatin was decross-linked for 15 minutes at 65ºC and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research, Freiburg, Germany). FOXO1 binding to Erag was assessed by quantifying ChIP-enriched DNA using CFX384 real-time PCR (Bio-Rad). Erag primers used: Erag-ChIP-forward (5’-TGCTGTGTAGCCTTTTGCAG-3’), Erag-ChIP-reverse (5’-TGGAAGCATAAAGCTGGTCA-3’). Luciferase assay Luciferase activity was measured using the Dual-Glo® Luciferase Assay System (Promega) according to the manufacturer’s protocol. Mouse WT (A70) and Rag2-/- (R2K3) cells were transfected (Fugene® 6 transfection reagent, Promega) with the Erag-Rag1-luciferase reporter (kindly provided by prof. M. Schlissel, University of Michigan, Ann Arbor, MI) and TK Renilla luciferase reporter vector (Promega) in the ratio 1:8 using in total 1.25 mg DNA per 100,000 cells. Following transfection cells were treated either with 5 mM KU55933 or vehicle (DMSO, Sigma) for 24h. Cells were harvested into 100 ml of passive lysis buffer. Firefly luciferase and Renilla luciferase activity were measured on a GloMax 96 microplate luminometer (Promega) according to the manufacturer’s instructions. Renilla activity served as a control for transfection efficiency. Activity of RAG reporter for each sample was calculated as firefly/Renilla ratio, expressed as relative light units (RLU).
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