Fig. 4. Decreased Tyr and Tyrp1 contents in H₂O₂-challenged B16 melanoma cells. (A) Cells were challenged with H₂O₂ (0.5 mM, 20 minutes), allowed to recover for the times shown, trypsin-harvested and solubilized. Equal amounts of protein (30 µg/lane) were electrophoresed on 9% SDS-PAGE gels and transferred to PVDF membranes. The blots were probed with PEP1 (anti-Tyrp1, first panel) and PEP7 (anti-Tyr, second panel), and stained with a chemiluminescent substrate (Amersham Pharmacia Biotech, Buckinghamshire, UK). Comparable loading and transfer was ascertained by cutting the lower portion of the blot and staining for total protein with Amido Black (lower panel). Similar trends were obtained in three independent experiments. C, control. Separate controls are included for cells grown for 16 and 24 hours after the oxidative challenge, as a density-dependent increase in melanocyte differentiation markers is often observed (Hornyak et al., 2000). (B) Densitometric quantification of immunoblots. Blots were quantified in a laser densitometer. The results shown represent the relative abundance of Tyr (white bars) and Tyrp1 (black bars), with respect to controls collected at 1 (for time points up to 8 hours), 16 and 24 hours, and are the mean±s.d. for three experiments.