Fig. 1

Characterization of cAMP-GEF II ^−/− mice. a. Schematic diagram for wild-type, floxed, and knockout (KO) alleles of cAMP-GEF II. Floxed mice were generated by gene targeting using MS12 ES cells derived from the B6 strain, and KO mice were generated by expressing Cre recombinase in the germ cells of the floxed mice (arrow, locus of primer (P1, P2, and P3) for genomic PCR). b, Genomic PCR analysis of cAMP-GEF II gene deletion in cAMP-GEF II ^+/− (HT, heterozygous), cAMP-GEF II ^+/+ (WT, wild-type), and cAMP-GEF II ^−/− (KO, knockout) mice. c, Western blot analysis of cAMP-GEF II protein expression in fractionated brain lysates. cAMP-GEF II protein expression was compared among S1 (postnuclear), P2 (crude membrane), and SPM (synaptic plasma membrane) fractions. cAMP-GEF II protein was highly expressed in SPM fractions, which also presented high expression of PSD95. Note that cAMP-GEFs protein expression was completely abolished in the brain of cAMP-GEF II ^−/− mice. d, Immunohistochemical analysis of cAMP-GEF II expression in brain tissue sections. Strong immunolabeling was observed in the cortex and hippocampus of WT mice, but was absent in KO mice. In the hippocampus, immunoreactivity for cAMP-GEF II was relatively low in the stratum pyramidale (sp) of the Cornu Ammonis (CA) as well as in the granular cell layer (gcl) of the dentate gyrus; while the stratum oriens (so), radiatum (sr), and lacunosum moleculare (sl-m), as well as the molecular layer (ml) of the dentate gyrus showed strong immunoreactivity for cAMP-GEF II. e, Immunofluorescence for NeuN showed that there was no difference in morphology of the hippocampus between the two genotypes. Scale bars = 500 μm in D, E. Abbreviations: SM, size marker