DNA binding activity of wild-type and mutant (Ser¹⁶⁹ → Ala) p45 in BHK cells. BHK cells were cotransfected with increasing amounts of expression vector encoding wild-type p45 [lanes 1 to 3, 0.3 μg, 0.6 μg, and 1.2 μg of pMT2-p45(wt), respectively] or mutant (Ser¹⁶⁹ → Ala)p45 [lanes 4 to 6, 0.3 μg, 0.6 μg and 1.2 μg of pMT2-p45(mut), respectively] and equimolar amounts of p18 expression vector [pMT2-p18]; lanes 7 and 8 show mock-transfected cells. Ten micrograms of nuclear extract protein was incubated with 10 fmol of end-labeled oligodNT probe encoding a NF-E2 recognition site (A) or a SP-1 recognition site (B); lane 9 shows probe incubated in the absence of nuclear extract protein. EMSAs were performed as described in Materials and Methods. Nuclear extracts incubated with the NF-E2 oligodNT probe yielded 2 protein/DNA complexes (A, lanes 1 to 6); both were eliminated by adding a 50-fold excess of unlabeled oligodNT, but only the slower migrating complex was eliminated by adding excess unlabeled oligodNT containing a mutation that abolishes NF-E2 binding, but not AP-1 binding (data not shown).18 The arrow in (A) indicates the protein/DNA complex containing p45 and p18 NF-E2.