pS647-Drebrin is negatively regulated by PTEN in neurons independently of PI3K.

<p>(A) Cortical neurons isolated from <i>PTEN</i>^{<i>flox/flox</i>} mice were transduced with increasing concentrations of Cre-expressing viruses at 13 DIV. Neuronal cell lysates were analysed at 19 DIV. Bar graph represents the relative band density of pS647-DBN and PTEN in three independent experiments with maximal cre-virus titer used, <u>+</u> sem. *p<0.05, *** < 0.001. (B) Rat cortical neurons were treated with the PI3K inhibitor LY294002 at indicated concentrations for 60 minutes before analyses of neuronal cell lysates. Bar graph represents the average band density of pS647-DBN in three independent experiments. (C) Hippocampal neurons were cultured for 1.5 DIV and labelled with anti-DBN, anti-pS647-DBN and anti-PTEN antibodies. (D) 18 DIV hippocampal neurons were stained with anti-DBN, anti-pS647-DBN and anti-PTEN. Top images show a specimen images at higher magnification. DBN is highly enriched in dendritic spines, but also found - albeit at lower levels - in the dendrite and the soma, whereas phosphorylation at S657 is more confined to the dendritic spine compartment. (E) Hippocampal neurons were cultured for 18 DIV and stained with anti-pS647-DBN and anti-PTEN antibodies. In dendritic spines (<b>arrows</b>), DBN is highly phosphorylated on pS647, whereas PTEN is mainly present in the dendrite process. Sporadically, PTEN is found in spines (<b>star</b>), which coincides with depleted pS647-Drebrin labeling. (F) Cortical neurons isolated from PTEN floxed/floxed mice were nucleofected with RFP or Cre-RFP and cultured for DIV3 before staining for pS647-DBN. Scale bars C, D, F: 10 µm; E: 2 µm.