Figure 3

Run-on experiment. A. Dot blot. 1, 2 and 3 indicate the transfections with pFull-MBg, pFull and pGL3Promoter, respectively. In the experiments shown in lanes 1, 2 and 3, the nylon membrane has been spotted with 2 μg of pGL3Basic and in the experiment shown in lane 4, it has been spotted 2 μg of pBluescript to measure the background of the hybridization with the labelled nuclei of the cell lines transfected with pGL3 Promoter. * indicates that the exposure time is of about 2 hours whereas in 1, 2 and 4 the blots have been exposed over-night. B. Each dot blot has been counted in the β-counter and the cpm are reported. Bkg stays for background and indicates the cpm of the experiments shown in lane 4. C. As transfection normaliser we have co-transfected, in all the experiments, 200 ng of pCMV-Seap2 vector. The Seap activity was measured with SEAP Reporter Gene Assay (Roche). Since the transfection efficiency in NIH3T3 is about three times higher than in MeWo cells (see the Seap2 activity in section C), we have used, in the run-on experiments, one third of the purified NIH3T3 nuclei and all the nuclei purified from MeWo.