Fig. 5

DNA Binding is Required for the Oct6-Mediated Inhibition of HPV18 Replication (A) U2OS cells were transfected with the appropriate expression vectors. WCEs were prepared 2 days after transfection. Equal amounts of WCEs were incubated with 32-P-labelled double-strand oligonucleotides for 30 min and resolved on native 4% PAAG. (B) WT Oct6, Oct6-N-197, and Oct6-WF > CS proteins were overexpressed in U2OS cells. WCEs were analysed using immunoblotting and anti-Oct6 or GAPDH antibodies 2 days post-transfection. (C) U2OS cells were cotransfected with the HPV18-Nluc genome and expression constructs encoding either WT Oct6 or its mutants, Oct6-N197 and Oct6-WF > CS. An empty vector was used as a control. Nluc activity was measured 2 and 3 days post-transfection and normalized to that of AP. Normalized Nluc activity in the control cells was set as 1, and data of other samples were calculated relative to the control. Data are shown as an average mean ± SD, n = 3, ***- p < 0.001. (D) U2OS cells were cotransfected with the HPV18 genome and either an empty vector or Oct6 expression constructs. Total DNA was isolated 2, 3, and 4 days after transfection, treated with the BglI and DpnI restriction enzymes to linearize the HPV18 genome and digest the input DNA, respectively, and analysed using SB