Figure 3

PS-mediated neuroprotection requires Akt phosphorylation of FKHRL1. (A) Western blots for FKHR, FKHRL1 and AFX in nuclear fractions from mouse cortical neurons treated with tPA/NMDA for 4 h in the presence or absence of PS. (B) Signals for FKHR, FKHRL1 and AFX were normalized to histone H1. (C) Western blots for pFKHRL1 in whole-cell extracts from mouse cortical neurons prepared 4 h after tPA/NMDA treatment in the presence or absence of PS. Intensity of pFKHRL1 signal was measured by scanning densitometry and normalized to β-actin. (D) Time-dependent survival (12-72 h) of neurons after transduction with Ad. Akt^K179A. Cell survival was quantified with a WST assay. (E) Western blots for pFKHRL1 in whole-cell extracts prepared from neurons transduced with Ad.Akt^K179A or Ad.GFP and exposed for 4 h to tPA/NMDA in the presence or absence of PS. Intensity of the pFKHRL1 signal was normalized to β-actin. (F) Western blots for FasL in whole-cell extracts from neurons transduced with Ad.Akt^K179A or Ad.GFP and treated for 6 h with tPA/NMDA in the presence or absence of PS. Intensity of the FasL signal was normalized to β-actin. (G) Caspase-8 activity in neurons transduced with Ad.Akt^K179A or Ad.GFP and exposed to tPA/NMDA for 8 h in the presence or absence of PS. (H) Cell survival of neurons transduced with Ad.Akt^K179A or control Ad.GFP and exposed to tPA/NMDA for 24 h in the presence or absence of PS. In all studies murine PS was used at 100 nM and was added simultaneously with tPA/NMDA. Mean ± SEM, n = 3 independent cultures in triplicate. NS, non-significant.