6 DNA damage-induced p53 downregulates expression of RAG1 | 159 The formation of coding joints was assessed by qualitative PCR analysis of Vκ6-23 to Jκ1 rearrangement using previously published primers7. The PCR products were separated in 2% agarose gel containing ethidium bromide. Exogenous DNA damage and p53 induction DNA damage was induced by irradiating the cells ([137Cs], 5 Gy, 0.50 Gy/min) at a dose of 5 Gy. In some experiments, dose dependency was studied, in these cases, where indicated so, also a 0.5 Gy dose was applied. After g-irradiation, the cells were allowed to recover for 2 h at 37°C in an atmosphere of 5% CO2 and subsequently harvested. To study the effect on p53 activation without the genotoxic element, the cells were treated with Nutlin-3 by adding 2.5mM of the compound into the cell culture and after 2h the cells were thoroughly washed and collected. Western Blot The protein pellets were homogenized passing through a 27G needle, and protein concentrations were measured using the bicinchoninic acid assay (Sigma-Aldrich). For each sample, 15 μg of protein lysate was used for protein separation in Precise 4–20% gradient Tris-SDS gels (Thermo Fisher Scientific). Subsequently, separated protein lysates were transferred onto polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore, Burlington, MA); 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 h. Primary antibodies (Ab) were incubated overnight at 4°C. After a series of thorough washes with TBS-T, membranes were incubated with secondary Abs (goat anti-rabbit HRP or rabbit anti-mouse HRP; DAKO, Agilent Technologies, Heverlee, Belgium) for 2h at room temperature. Ab binding (protein expression) was visualized using Amersham ECL Prime Western blotting detection reagent (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The following Abs were used in this study: rabbit anti-RAG1 (D36B3), rabbit anti-FOXP1, rabbit anti–phospho-p53 (Ser15) (9284), rabbit anti-cleaved caspase-3 (Asp175) (9661), rabbit anti-BCL6, rabbit anti- KU70 (D10A7), all from Cell Signaling Technology, Danvers, MA, rabbit anti-FOXP1 ab16645 (Abcam), and mouse anti–β-actin (C4) (EMD Millipore). Real-Time quantitative PCR The RNA isolation from the cells was performed using TRIreagent (Sigma-Aldrich) according to the manufacturer’s protocol. MicroRNA isolation was performed using mirVana miRNA isolation kit (ThermoFisher) according to the manufacturer’s protocol. Levels of mRNA expression were determined using the CFX384 (Bio-Rad, Hercules, CA) real-time PCR platform. For each sample, cDNA was synthesized from 500 ng of RNA, using random primers (Promega) and Moloney murine leukemia virus reverse transcriptase (Invitrogen-Life Technologies, Bleiswijk, the Netherlands). For each sample, cDNA was
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