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. 1997 Jun 10;94(12):6450–6455. doi: 10.1073/pnas.94.12.6450

Figure 2.

Figure 2

107/402-T expression and episome copy number in transiently transfected HT-1376 cells. (A) At the indicated times (days 1–6), cell extracts were prepared for Western blot analysis of 107/402-T expression using anti-T monoclonal antibody 416. The 96-kDa 107/402-T protein is indicated by the arrowhead. Positive controls for T antigen expression include COS-7 and a clone of 5637 bladder carcinoma cells stably expressing 107-T (F9). (B and C) Southern blot analysis of HT-1376 cells transiently transfected with episomes encoding wild-type T or 107/402-T, respectively. At the indicated times (days 1–10), cells were harvested, and total cellular DNA (4 μg) was digested with both ApaI and DpnI. Linearized DNA (arrowhead) indicates the presence of replicated episome. A standard curve ranging from 20 to 20,000 copies per cell was calculated based on transfection efficiency and modal chromosome number. In B, standard curve plasmid DNA was linearized with ApaI, whereas uncut DNA was loaded in C. CMV, negative control pRC/CMV transfectants 3 days after gene transfer; DC, DpnI digestion control consisting of 4 μg of HT-1376 DNA and 2 ng of pRC/CMV.107/402-T.