Next, in separate experiments, we transfected the HLR cell line with the fusion trans-activator pFA2-CREB, pFA2-Elk1, or pFA2-cJun plasmids. After performing successive rounds of selection with G418, we chose the clones that gave the best response when transfected with the positive control vectors from the PathDetect kits. These clones were then subjected to more extensive analysis.
Genomic DNA from each stable clone was prepared and used as a template for PCR analysis to confirm that each fusion trans-activator plasmid was stably incorporated into the genomes of the selected clones. To amplify the activation domain for each reporter cell line, we used one primer specific to the GAL4 DNA-binding domain and another primer specific to each of the activation domains. Each primer set successfully amplified a product of the expected size. Analysis of HLR-CREB and HLR-Elk1 cell lines are shown in Figure 2. To further confirm the activity and specificity of these cell lines, we transfected each cell line with plasmids encoding known activators of each pathway. Upon luciferase assay of cell lysates, each cell line showed significant luciferase expression above background (Figure 3).
Additional testing included an analysis of the activation of luciferase
activity in these stable cell lines by extracellular stimuli. For example,
HLR-Elk1 cells were treated with known MAPK activators, including epidermal
growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) (Figure
4). Luciferase expression in the HLR-Elk1 cells wa