FIG. 9.

Mutation at serine 131 affects DNA-PK-mediated phosphorylation of Sp1. Sp1Gln, Sp1GlnM2, and Sp1ΔGln proteins were expressed and purified as GST fusion proteins. (A) Purified proteins stained by Coomassie brilliant blue. Arrow points to the migration position of Sp1Gln and Sp1GlnM2. Sp1ΔGln (lane 3) migrates with an apparent size that is approximately 5 kDa smaller than either Sp1Gln (lane 1) or Sp1GlnM2 (lane 2). (B) DNA-PK (purified enzyme purchased from Promega)-mediated transfer of ³²P from [γ-³²P]ATP to Sp1Gln (lane 1), Sp1GlnM2 (lane 2), or Sp1ΔGln (lane 3) in the absence (left) or presence (right) of double-stranded oligonucleotides. Arrow points to phosphorylated Sp1Gln protein seen only in the reaction supplemented with DNA (lane 1, right).