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. 2003 Nov;77(21):11425–11435. doi: 10.1128/JVI.77.21.11425-11435.2003

FIG. 3.

FIG. 3.

Sp1/3 binds to the ORF50 promoter in vitro and in vivo. (A) BCBL-1-derived nuclear extracts from NaB-treated (+) or untreated (−) cells were subject to DNA affinity purification with DNA derived from pBKSII (BKS), ORF50 (−298/+1), or the GC-box substitution mutant of ORF50 (−114) (ORF50mt). Bound proteins were assayed by Western blotting of SDS-polyacrylamide gel electrophoresis gels with antibodies specific for Sp1 (αSp1), Sp3 (αSp3), or Rb (αRb) as indicated to the left of each panel. (B) NaB-treated (+) or untreated (−) cells were analyzed by ChIP with antibodies specific for Sp1 or control IgG. Promoter fragments from the ORF50 promoter (left panel) or for the coding sequences of GAPDH (right panel) were generated by PCR. Input DNA is indicated in the lower panel. Threefold dilutions of input DNA were used to determine the linearity of PCRs. (C) 293 cells transfected with ORF50-Luc plasmid were assayed by ChIP for association with Sp1 before and after NaB treatment.