Figure 3.
Dasatinib inhibits nuclear translocation of ERK1/2 in Mel-p. (A) The levels of phospho-ERK1/2Thr202/Tyr204 and total ERK1/2 were assessed in Mel-p cells treated with various concentrations of dasatinib for 24 h using western blot analysis. (B) Location of activated ERK1/2 by immunofluorescence microscopy. Mel-p cells were plated on glass coverslips in the presence or absence of dasatinib for 24 h, serum starved overnight and stimulated with 10% fetal bovine serum (FBS). At the indicated times, cells were fixed with 60% acetone/3.7% formaldehyde, stained with rabbit anti-phospho-ERK1/2Thr202/Tyr204 (green), and nuclei were stained with DAPI (blue). As shown in the upper panel, in control cells, nuclear localized p-ERK was detectable within 5 min and reached a maximum by 30 min, whereas the pERK1/2 in dasatinib-treated cells was mainly cytoplasmic at all times after serum addition. (C) Schematic depicting regulation of nucleocytoplasmic MAPK activity by dasatinib. The nuclear activity of ERK is required for mitogen-stimulated cell proliferation and dasatinib inhibits the nuclear translocation of ERK signaling. MAPK, mitogen-activated protein kinase.