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, Alexa Fluor 488-labeled antirat and Alexa Fluor 546-labeled anti-mouse

, Invitrogen) for 1 h at RT, cells were again washed with PBS, counterstained with 2 µg/ml DAPI, and washed and fixed with Vectashield antifade mounting medium (Vector Laboratories). Images were taken at RT with a confocal laser-scanning microscope

, M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF during a 30-min incubation at 4°C. Nuclear extracts were centrifuged to obtain the solubilized nuclear fraction. the affinity-purified antiserum was confirmed on extracts from 293T cells overexpressing mPIDD alone or together with an mPIDD-specific short hairpin RNA construct (Tinel and Tschopp, 2004) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instruction. Lysates were generated 24 h after transfection (see Biochemical fractionation and immunoblot analysis) and subjected to SDS-PAGE separation and immunoblotting. Lysates from MEFs stably transfected with mPIDD served as a positive control. Whole body irradiation experiments PIDD-deficient mice and littermate controls, .) equipped with LSM 510 acquisition software version 2.8 (Carl Zeiss, Inc.) and a 63× NA 1.4 oil immersion lens at a resolution of 1024 × 1024 pixels, with pinholes set to acquire images <1 µm thick. The adjustment of brightness and contrast to enhance visibility of details was performed using the LSM Image Browser software version, vol.4

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