Fig. 3. S1P induces the degradation of MITF and stimulates the ERK signaling pathway. (A) After serum starvation, Mel-Ab cells were stimulated with 10 µM of S1P at the times indicated. Whole cell lysates were then subjected to western blot analysis with antibodies against MITF, phospho-specific ERK, and phospho-specific MEK. Equal protein loading was checked by reaction with actin, phosphorylation-independent ERK, and MEK antibodies. (B) RT-PCR analysis of MITF mRNA levels in S1P-stimulated cells. Cells were treated with S1P as in A. Total RNA was isolated from the cells and cDNA prepared. Equivalent amounts of cDNA were amplified with primers specific for MITF, and actin primers were used as a control to ensure the even loading of target cDNA. The resulting PCR products were analyzed by agarose gel electrophoresis. The lane on the left shows markers of the indicated size. NC, negative control.