PP2A mediates dephosphorylation of ezrin by cisplatin. Cells plated on 10-cm dishes (5 × 105 cells/dish) were pretreated with 10 nM of either okadaic acid (A) or tautomycin (B). After 1 h, cells were incubated with DMSO or cisplatin (5 μg/ml) for 1 h. Phospho-ezrin levels were evaluated by Western blotting using a specific polyclonal antibody. (C) Cells were transfected with 20 nM of either SCR or PP2A (catalytic subunit) specific oligonucleotides. After 48 h, cells were subjected to DMSO or cisplatin treatments for 1 h. Lysates (30 μg) were analyzed by Western blotting for levels of p-ezrin and PP2A. Blots shown are representative of at least three independent experiments. Densitometric analysis was performed using NIH Image software. (D) MCF-7 cells expressing YFP-ezrin and CFP-PP2A (catalytic subunit) were subjected to sensitized emission FRET analysis. After 24 h of plasmid transfection, cells were treated with DMSO or cisplatin for 30 and 60 min. A representative FRET image of at least 30 cells imaged in three experiments is shown. FRET efficiencies are encoded by using the color bar scale shown on the left. Colors range between blue (lowest FRET) and red (highest FRET).
