Role of BMP-4 and Its Signaling Pathways in Cultured Human Melanocytes
Figure 3
Effects of BMP-4 on the level of phosphorylated level of MITF. (a) Paired cultures of melanocytes were treated with BMP-4 (25 ng/mL) for 0, 0.5, 1, 2, and 5 hours. At each time point, cells were harvested and immunoblot analysis using monoclonal antibody against MITF was performed (provided by Dr. David Fisher from the Dana Farber Cancer Institute). This antibody has been shown to recognize both phosphorylated and nonphosphorylated MITF. As the loading control, the membrane was immunoblotted for actin. One representative result from three separate experiments is shown. (b) Paired cultures of melanocytes were treated with BMP-4 (25 ng/mL) for 15 and 30 minutes in presence or absence of MEK inhibitor PD98509 at 10 m. At each time point, cells were harvested and the level of MITF protein was determined. One representative result from three separate experiments is shown. (c) Paired cultures of melanocytes were treated with BMP-4 (25 ng/mL) and cycloheximide (15 ug/mL) for 0, 0.5, 1, 2, and 5 hours in presence or absence of proteosome inhibitor MG-132 at 10 m. At each time point, cells were harvested and the level of MITF protein was determined using immunoblot analysis. One representative result from three separate experiments is shown.
