Figure 2. Schematic representation of phsp70-2R-CAT. Panel A: The parent plasmid hsp-cat 1 was digested at a unique XhoI site, 195 nucleotides upstream of the transcription start site of the CAT reporter gene. The insert labeled 2R was produced using primers EcRE-1 and EcRE -2 (Fig. 1), digested with SalI and XhoI, and ligated to the XhoI digested hsp-cat 1 plasmid. Panel B: Heat-shock inducible CAT activity from phsp70-2R-CAT. Cells were treated with 10-5M 20E, 24 h after transfection. Heat shock included a 4 h treatment at 41°C, followed by a 2 h recovery, immediately before the cells were harvested and lysed. The CAT assay was done as described previously (Durbin and Fallon, 1985), but the incubation time was extended to 12 h.