Figure 3.

PPZ dephosphorylates Akt in a PP2A-dependent manner and dephosphorylates ERK1/2 with no dependency on PP2A. The phosphorylation of Akt and ERK1/2 in (A) HUT78 and (B) UL-1 cells were determined via western blotting. Representative images from three independent experiments are presented. The effect of PPZ (10 µM) on the activity of recombinant MEK1/2 was analyzed by performing an in vitro kinase assay with recombinant ERK1/2 as a substrate. Phosphorylation levels of ERK1/2 were detected via (C) western blotting, with subsequent (D) quantification from three independent experiments. (E) HUT78 cells were pre-incubated with tautomycin (1 µM), cyclosporin A (10 µM) and sanguinarine (10 µM) for 4 h, as well as with BCI (10 µM) for 1 h. Then cells were treated with PPZ (20 µM) for 15 and 30 min. The phosphorylation of ERK1/2 was detected by western blotting. Representative images from two to three independent experiments are presented. HUT78 cells, following pre-incubation with Na₃VO₄ (300 µM) for 1 h, were treated with PPZ (20 µM) for 15 and 30 min. (F) Representative images and (G) quantitative data from three independent experiments were presented. *P<0.05 as indicated. PPZ, perphenazine; PP2A, protein phosphatase 2A; Ctrl, control; VCP, valosin-containing protein; p, phosphorylated; OA, okadaic acid.