Reduced PKMζ expression by inhibition of Notch signaling. (A) Western blot analysis of glutamate receptor subunits and several proteins. Hippocampal lysates were prepared from 3 wild-type and 3 mib1 cKO mice at 6 months of age, then fractionated and subjected to immunoblotting. Note that PKMζ protein is significantly reduced in the total and synaptosomal fractions of mib1 cKO hippocampal lysate. (B) Hippocampal lysate from 6-month-old wild-type and mib1 cKO mice were analyzed using an in vitro Cdk5 kinase assay to determine the autoradiography using histone H1 as a substrate. (C) Reduced total PKMζ protein in the hippocampus of mib1 cKO mice (n = 5) compared to wild-type mice (n = 5). (D) Total pkmζ RNA from 6-month-old wild-type (n = 6) and mib1 cKO (n = 6) hippocampi were analyzed by quantitative RT-PCR. (E) Reduced pkmζ mRNA transcription by inhibition of Notch signaling in primary hippocampal neurons. DIV14 primary hippocampal neurons were treated with 20 μM DAPT for 12 h, and total RNA was analyzed by quantitative RT-PCR. (F) Reduced PKMζ protein expression by DAPT treatment. DIV14 primary hippocampal neurons were treated with 20 μM DAPT for 24 h and subjected to immunocytochemistry with MAP2 and PKMζ antibody. DMSO-treated cells showed robust expression of PKMζ protein in the cell body and dendrites. DAPT-treated cells showed decreased expression of PKMζ protein. Arrows in lower panels indicate PKMζ protein in dendrites. Scale bars: 50 μm in upper and middle panels; 20 μm in lower panels. (G) Reduced PKMζ protein expression by DN-MAML-GFP transfection. DIV10 primary hippocampal neurons were transfected with EGFP and DN-MAML-GFP DNA and subjected to immunocytochemistry on DIV14. EGFP immunoreactivity was used to identify transfected cells. EGFP transfected cells showed robust expression of PKMζ protein in the cell body and dendrites. DN-MAML-GFP transfected cells showed reduced expression of PKMζ protein, especially in dendrites. Note that nontransfected cells show significant expression of PKMζ protein. Scale bars: 20 μm. Error bars show standard deviation. *Significant difference; p < 0.01. **Significant difference; p < 0.001.