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, To permeabilize the cells, PFA was replaced with 70% prechilled ethanol for 5 min at RT. After three washes with PBS, unspecific binding sites were blocked by incubation with 8% BSA (in PBS) for 1 h at RT. Incubation with primary antibodies, with 2% PFA at RT for 20 min
both in blocking solution) was performed at 4°C overnight followed by three washings with PBS for 10 min each. After incubation with secondary antibodies (1:100 in blocking solution ,
, 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
, Immunofluorescence staining, flow cytometric analysis, and cell sorting Single-cell suspensions from peripheral blood, bone marrow, lymph nodes, spleen, and thymus were surface stained with monoclonal antibodies conjugated with FITC, R-phycoerythrin (PE), allophycocyanin, or biotin (Invitrogen). The monoclonal antibodies used and their specificities are RA3-6B2
, GK1, vol.5, p.4
,
, Ter119, anti-erythroid cell surface marker; T24.31.2, anti-Thy-1
, H57-59, anti-TCR-? (all from eBioscience)
,
, Clonal survival of MEFs Colony formation of primary or SV40-immortalized MEFs was assessed by seeding an increasing numbers of cells per well (6,000, 12,000, and 18,000 cells) 24 h before DNA damage or exposure to heat shock. To induce heat shock, cells were incubated for 60 min in a tissue culture CO 2 incubator (CB150; Binder) set to 43°C or 45°C and then grown under standard conditions. Etoposide was removed after 24 h of incubation, anti-CD23 (both from BD). Biotinylated antibodies were detected using streptavidin-R-PE (Dako) or streptavidin-vPE-Cy7 (BD), vol.3, p.303
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