KIT activation up-regulates MITF protein expression. (A) Western blot shows robust increase of MITF protein expression by 24 to 48 hours in primary murine BMMCs treated with SCF (100 ng/mL). Relative expression is calculated by taking the ratio of the densitometry signal for MITF to tubulin and normalizing to 1.0 for the zero time point. (B) Real-time PCR shows MITF mRNA levels with SCF treatment in BMMCs. Signal quantitated with SYBR Green and normalized to β-actin. Relative expression is normalized to the signal of the zero time point. (C) Pharmacologic inhibition of KIT reduces MITF expression. Western blot shows that increasing the concentrations of the KIT inhibitor imatinib results in a dose-dependent decrease in MITF expression in murine (P815). Relative expression is calculated as described for BMMCs. (D) Real-time PCR shows no significant change in MITF mRNA in murine mastocytoma (P815) with imatinib treatment (10μM). (E) MITF protein half-life does not change significantly with KIT activation. Metabolically labeled BMMCs (primary mast cells) with and without SCF treatment were immunoprecipitated with an MITF antibody, and MITF protein was quantitated at various time points after SCF treatment with densitometry of signal intensity of bands on autoradiograph. There was no significant difference in MITF half-life with SCF treatment.