FIG. 4.

(A) HPV8 E2 binds to specific sites within the β4-integrin promoter with different affinities. ³²P-labeled oligonucleotides comprising a single E2 binding site (BS-II and BS-III) were incubated with 28 μg of nuclear extracts isolated from pEYFP-C1- and pEYFP-HPV8-E2C-transfected 293T cells. In reaction mixtures containing BS-I, fivefold more nuclear extract was added. Oligonucleotides comprising the first E2 binding site (control E2-BS) within the HPV18 long control region served as controls. The arrows indicate binding activities appearing in the EYFP-8E2C-containing extracts. (B) Supershift analysis of the EYFP-8E2C binding activity on BS-II. Serial dilutions (1:5, 1:10, 1:20, and 1:40) of antibodies directed against the EYFP moiety were added to the nuclear extracts isolated from 293T cells transfected with pEYFP-HPV8-E2C prior to the addition of ³²P-labeled wild-type BS-II oligonucleotides. Supershifted complexes are indicated by an asterisk. Arrows indicate the EYFP-HPV8-E2C specific bands, which disappeared with increasing doses of anti-EYFP antibody.