Figure 5

PS neuroprotection requires Tyro3 activation. (A) Cell survival of cortical neurons isolated from Tyro3, Axl or Mer null mice and wild type (wt) mice after 24 h of tPA/NMDA exposure in the presence or absence of PS. Cell survival was quantified with a WST-8 assay. (B) PS dose-dependently mediates Tyro3 tyrosine phosphorylation in tPA/NMDA-treated mouse cortical neurons as determined by immunoprecipitation (IP) with anti-phospho-tyrosine (pTyr) antibody followed by immunoblotting (IB) with anti-Tyro3 antibody and anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody (as a loading control) 2 h after tPA/NMDA. Graph, relative abundance of phosphorylated Tyro3 to VEGFR2. (C) Akt phosphorylation (pAkt, Ser473) in neurons isolated from Tyro3 null mice and wt controls exposed to tPA/NMDA for 2 h in the presence or absence of PS. (D) FKHRL1 phosphorylation (pFKHRL1, Ser253) in neurons isolated from Tyro3 null mice and wt controls exposed to tPA/NMDA for 4 h in the presence or absence of PS. In all studies murine PS was used at 100 nM unless specified and was added simultaneously with tPA/NMDA. Mean ± SEM, n = 3 independent cultures in triplicate. NS, non-significant.