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, The proteins were transferred onto nitrocellulose membranes (Amersham Protran) for Western blot analysis. Following transfer, membranes were rinsed in PBS and blocked in PBST (PBS plus 0.1% Tween 20) + 2% skimmed milk from Difco for one hour at room temperature. The membranes were then incubated overnight at 4 °C in PBST containing a 1:5000 dilution of either a monoclonal anti-V5 antibody, Western blot analysis. Extracted or immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis on NuPAGE R Novex Bis-Tricine 4-12% or Tris-Glycine 10-20% pre-cast gels

, All PCR products were purified from agarose gel with the QiaQuick gel extraction kit (Qiagen) to remove residual primers. A second PCR amplification was performed with P, T and the URA3 or LEU2 marker with the following program: 2 min at 94 °C, then 5 cycles of 30 s at 94 °C, 30 s at 60 °C, 1.5 min at 72 °C, followed by 25 cycles of 30 s at 94 °C, 30 s at 60 °C, 3 min at 72 °C. Primer pairs 23-28 or 31-36 were added after the 5 first cycles. The resulting PCR products were purified from agarose gel and about 400 ng of the purified cassettes was used to transform ?ku70. Transformants were selected on YNB complemented with histidine and leucine or uracil depending on the marker used, Gene deletion. KU70 (YALI0C08701g) deletion was performed using the deletion cassette ylKU70 PUT purified on agarose gel (QiaQuick gel extraction kit, Qiagen, Courtaboeuf, France) from NotI-digested plasmid JME1264 70

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