Fig. 4. Melanogenic protein and gene expression and Erk hyperactivation in Nf1^+/– primary melanocytes. (A) Melanogenic protein expression in mixed primary culture wild-type and Nf1^+/– cells. Lysates were prepared on day 10 of primary culture with TPA, bFGF and dbcAMP withdrawn 72 hours prior to lysis. Western blotting shows relative expression of p-Erk, Tyr, Tyrp1, Mitf and Dct/TRP-2 as indicated. Expression of Erk2 is used as a loading control. (B) Melanogenic gene expression in mixed primary culture wild-type and Nf1^+/– cells. Total RNA was prepared on day 10 of primary wild-type and Nf1^+/– cultures. Relative expression of Tyrp1, Dct, and Tyr was determined by real time RT-PCR. Data is representative of results from two independent experiments. Error bars represent the standard deviation from the mean of duplicate samples. (C) SCF/Kit-ligand-dependent and SCF/Kit-ligand-independent Erk phosphorylation in wild-type and Nf1^+/– cells. Mixed wild-type and Nf1^+/– primary cultures were grown for 15 days. TPA, bFGF and dbcAMP were withdrawn 72 hours prior to stimulation with murine SCF for 5 minutes and 15 minutes. Cell lysates were collected and examined for relative expression of phosphorylated Erk and Erk2 by western blotting.