Fig. 8

Dexras1-mediated iron flux modulates phosphorylation of NR2A via PKC-Src pathway. a. Phosphorylated Src is increased following treatment with PIH. Brain slices from WT mice were treated with 50 μM PIH for 15 min and various proteins were quantified via western blot analysis. ARC, an immediate early gene and important signaling molecule in synaptic plasticity, is elevated as well as pSrc but not unphosphorylated total Src. The bottom band is the internal control GAPDH. b. Western blot analysis of brain homogenates show that there are differences in NR2A protein expression between WT and Dexras1 knockout mice and that there is a decrease in the baseline level of phosphorylated pSrc (n = 4). PIH application does not induce phosphorylation of Src. The bottom band is the internal control GAPDH. c. Hippocampal slices were pre-treated with calphostin C (10 μM) for 15 min and followed by 15 min PIH treatment. Western blot analysis show that calphostin C treatment blocks PIH-mediated activation of pSrc and NR2A. The bottom band is the internal control GAPDH. d. Hippocampal slices were pre-treated with either L-NAME (1 mM) or Ebselen (10 uM) for 15 min as described in Fig. 6 and followed by 15 min PIH treatment. Western blot analysis show that these treatments blocks PIH-mediated activation of pSrc and NR2A. The bottom band is the internal control GAPDH. e. Iron chelatrion increased PKC activity while inhibition of nNOS or DMT1 blocked PIH-mediated activation of PKC. Brain slices from WT mice were pretreated with either L-NAME or Ebselen and subsequently treated with PIH as described above. Tissues were lysed and PKC activity was measured