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. 1999 Dec;73(12):10146–10157. doi: 10.1128/jvi.73.12.10146-10157.1999

FIG. 1.

FIG. 1

T-antigen induces binding of YB-1 to the 23-bp early strand (23E) in electrophoretic mobility shift assays. (A) Interaction of bacterially expressed MBP, MBP–YB-1 fusion protein, and baculovirus-expressed T-antigen with the 23-bp early single-stranded DNA probe. The 23E probe was incubated with 30, 60, or 240 ng of MBP alone (lanes 2 to 4, respectively) or MBP–YB-1 fusion protein (lanes 5 to 7, respectively) or with 100, 200, or 400 ng of T-antigen (T-Ag) (lanes 8 to 10, respectively). DNA-protein complexes were resolved on a 6% polyacrylamide gel under native conditions. Positions of MBP–YB-1 and T-antigen complexes with the 23E probe are indicated by arrows. (B) Competitive band shift assay. The 23E probe was incubated with MBP–YB-1 protein in the absence (lane 1) or presence of a 50- or 250-fold molar excess of unlabeled competitor DNA as indicated. 23E represents the 23-bp wild-type early single-stranded oligonucleotide (5′-CAGTTTTAGCCAGCTCCTCCCTA-3′) (lanes 2 and 3), mut 23E represents a mutant 23E single-stranded oligonucleotide (5′-AGTATTACACAGATATTTATTAC-3′) (lanes 4 and 5), and ds23 represents the wild-type double-stranded 23-bpse (lanes 6 and 7). (C) T-antigen induces binding of YB-1 to the 23-bp early single strand. The 23E probe was incubated with recombinant YB-1 (MBP–YB-1), either alone (lane 1) or with baculovirus-expressed T-antigen (lanes 2 and 3), or with T-antigen alone (lane 4). Formed DNA-protein complexes were resolved on a 6% polyacrylamide gel under native conditions. Respective protein concentrations used for the binding reactions are shown above the panel. The protein concentration used for all binding reactions was kept constant by addition of MBP. MBP–YB-1:23E and T-Ag:23E complexes are indicated by arrows. (D) Bacterially expressed, purified GST protein does not induce binding of YB-1 to the 23-bp early single strand. The 23-bp probe was incubated with recombinant YB-1, either alone (lane 1) or in combination with GST (lanes 2 and 3), or with GST alone (lane 4). Formed DNA-protein complexes were resolved as described for panel C. Respective protein concentrations used for the binding reactions are shown above the panel. MBP–YB-1:23E complex is indicated by an arrow. In all panels, brackets indicate free probe.