Similarly, when ZNF274 or KAP1 ChIPs were sequentially immunoprecipitated with SETDB1 antibodies, there is specific enrichment of the ZNF180 binding site (lanes 4 and 7, respectively).
Similarly, competing DNA mutated in a single site (lanes 5, 6, 7, 8) showed that band b corresponds to complexes on the TRE, while band a represents binding to the CRE.
As shown in Fig. 6E, under resting conditions, Sp1 binds to the I Sp1 (lane 1), II Sp1 (lane 3), and III Sp1 (lane 5) binding sites and NF-κB (p65) binds to the NF-κB-binding site (lane 7).
The nuclear extracts from either ARPE-19 cells or from FHL124 cells showed a strong binding to CREB site (lane 1, 6 of Fig. 4B), this binding can be competed off by the cold CREB oligo (lane 2, 7 of Fig. 4B) but not by the mutant oligo (lane 3, 8 of Fig. 4B).
It migrated slightly above the appropriate rung of a Maxam-Gilbert sequencing ladder derived from the labeled strand of the half-site (lane 1 (MG), Figure 6B), and co-migrated with a synthetic 24-mer of the predicted sequence bearing a 3'-OH (lane 3 (M), Figure 6B).
The nuclear extracts from either ARPE-19 cells or from FHL124 cells showed a strong binding to AP-2α (1) site (lane 1, 6 of Fig. 5B), this binding can be completely competed off by the cold AP-2α (1) oligo (lane 2, 7 of Fig. 5B) but only partially by the mutant oligo {lane 3, 8 of Fig. 5B, A change of 3-nucleatide in the AP-2α (1) binding site only partially abolished the binding activity}.
It is important to note that these levels are comparable to those from a parental construct, pAdCMV5-LacZ, lacking the operator site (lane 1).
Thus RT-PCR analysis shows that continuous CCNB1 pre-mRNA is detected up to ∼1.1 kb downstream of the poly(A) site (lane 8, F/R3 primer pair), with no continuous RNA detected beyond this point (lanes 9 and 10).
ULK3 binding also mapped to the MIM1 of CHMP1B as binding was inhibited by removal of CHMP1B MIM1 or by MIM1 point mutations, but not by a mutation in the more extended CHMP1B MIM3 binding site (lane 8) (Yang et al., 2008).
As shown in Figure 6, a prominent DNA protein complex was formed when nuclear proteins from Oli-neu cells were incubated with a positive control probe containing a single YY1 (consensus) binding site (lane 7).
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