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. 2011 May 31;6(5):e20586. doi: 10.1371/journal.pone.0020586

Figure 5. Caspase-3 mediates PP2A-related AKT dephosphorylation.

Figure 5

A. Different cells were pre-incubated with indicated concentrations of OA for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. β-Actin was used as a protein loading control. Relative amount of p-AKT and AKT in untreated cells were set as 1. B. Analysis of the effect of caspase-3 on PP2A activation. Up, Cells were pre-incubated with indicated concentrations of DEVD-CHO for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. Relative amount of p-AKT and AKT in untreated cells were set as 1. Middle, the treated cells were lysed with sample buffer and subjected to immunoblot assay with an A subunit-specific anti-PP2A/A antibody. CF is referred to cleaved PP2A/A. The membrane was then stripped and reprobed with a C subunit-specific anti-PP2A/C antibody. Relative amount of PP2A/C in untreated cells (0 h) were set as 1. Down, Western blot analysis of the effect of DEVD-CHO (100 µM) in PP2A cleavage. C. Detection of PP2A activity. Cells were pre-incubated with or without 0.2 µM of OA or 100 µM of DEVD-CHO for 1 h and exposed to drug for 48 h. PP2A activity with the cellular proteins extracted from RIPA buffer-lysed cells was measured using phosphopeptide KIpTIRR as a substrate as described in Materials and Methods. Each column represents the mean ± S.D. of triplicate assays. *, P<0.05. D. Detection of the binding of AKT with PP2A. Cells were incubated with or without ATO for 48 h. In some groups of cells, 0.2 µM of OA or 100 µM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with RIPA buffer for immunoprecipitation with anti-AKT antibody followed by immunoblot assay with anti-PP2A/C and anti-AKT antibodies. Relative amount of individual protein in untreated cells were set as 1. Data are representative of at least three independent experiments.