Figure 1

ATF-2 expression and phosphorylation in B16 malignant melanoma vs. non-malignant melan-a mouse melanocytes. A. Subcellular distribution and relative amount of ATF-2 in B16 mouse melanoma compared with nonmalignant melan-a cells. B16 cells and Melan-a cells were harvested at 80% confluence, and cytoplasmic and nuclear protein were isolated using Pierce NE-PER™ nuclear and cytoplasmic extraction reagents described in Materials and Methods. Cytoplasmic and nuclear proteins (10 μg) from both cell lines were analyzed by western blotting using polyclonal ATF-2 antibody. The autoradiogram (top right panel) was scanned using a Molecular Dynamics densitometer, and after correcting for the amount of β-actin, the relative amount of total ATF-2 in each sample was determined (bottom). The data shown are from a representative experiment, which was replicated three additional times with similar results. The top left panel illustrates the morphology of the two different cell lines prior to harvest (phase contrast, 20X). B. Phosphatase digestion of ATF-2. Nuclear extracts prepared from B16 cells were treated with 2 units of PP1 at 37°C. The reaction was stopped by the addition of SDS-sample buffer at the indicated incubation times. The samples were boiled, then blotted and detected using phospho-ATF-2 antibody. GAPDH was used as an internal control. The data are representative of three individual experiments with similar results. C. Relative amount of phospho- ATF-2 in B16 cells vs. melan-a cells. Cellular extracts (20 μg) from B16 cells and cellular extracts (40 μg) from melan-a cells (minimal amount of protein that allowed signal detection of phosphorylated ATF-2) were analyzed by western blotting using anti-ATF-2 polyclonal antibody and anti-phospho-ATF-2 polyclonal antibody, as described in Materials and Methods. The autoradiogram (top) was scanned using a Molecular Dynamics densitometer, and after correcting for the amount of total ATF-2 protein, the relative amount of phospho-ATF-2 protein in both cell lines was determined (bottom). The data shown are from a representative experiment, which was replicated three additional times with similar results.