Fig 4.

Immortalization of BRK cells by HPV E7 and E6. Lentiviral constructs that express HA-tagged E7 and Flag-tagged E6 (with an intervening internal ribosome entry site [IRES] sequence for E6 translation) were generated using the vector pCDH–MCS-EF1-Puro (System Biosciences). Lentiviral particles were generated by transfection of 293 T cells with the pCDH-HPV plasmids pVSVG and pCMVΔ8.2ΔVPR (39) using the Lipofectamine reagent. The BRK cells were infected with the virus for 2 to 4 h in the presence of Polybrene (8 μg/ml), and the cells were subjected to puromycin selection (0.25 μg/ml). When the cells reached about 80% confluence, they were split to 1:2, serially. The number of passages was plotted against weeks of propagation. For each construct, columns 1 to 5 show results of five independent immortalization assays. The expression of Flag-E6 proteins in immortalized BRK cell lines that express 5E6 wt or 5E6L21S is shown in the inset. The Flag-E6 proteins were immunoprecipitated with the Flag Ab, and the Western blots were probed with the same Ab.