FIG. 4.

Characterization of Rep68 binding to the E2a promoter region by EMSA. (A) The 303-bp promoter element from pE2aLUC was obtained by digestion with HindIII and XhoI and end labeled. The 162- and 141-bp promoter fragments were obtained by digestion of the 303-bp element with NciI. EMSA was performed by using ∼10 fmol of each DNA fragment with increasing amounts of Rep68 protein. (B) The 303-bp fragment was used as a template to amplify a 150-bp fragment containing sequences essentially identical to the 141-bp element in panel A. Primers corresponding to the ends of the 141-bp fragment (with an additional 9 nt) were used to amplify the 150-bp fragment in a reaction containing [α-³²P]dATP. EMSA was performed by using both affinity purified VP3 and Rep antibody. Totals of 250 and 500 ng of antibody were used. Antibodies contained ∼50 μg of IgG/ml. (C) Increasing amounts of Rep68 were added to 7.5 fmol of the 150-bp fragment. (D) The dissociation constant was determined after performing the titration in panel C in quadruplicate. The dried gel was exposed to a phosphorimager cassette, and densitometry was performed with ImageQuant software.