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Cells were transfected with constructs expressing Flag-tagged wild-type Pellino1 or Pellino1 S39A mutant. Wild-type or mutant Pellino1 was purified and incubated with 1 µg of recombinant GST-DAPK1 fusion protein in the presence of 0.2 mM ATP for 30 min. Reaction was terminated by addition of SDS-containing lysis buffer and the reaction products were resolved by SDS-PAGE and detected by western-blotting.
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