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Northern blot analysis. Total RNA was extracted from several Olmesartan regions (cortex, diencephalons, hippocampal formation, cerebellum and brainstem). RNA (20 μg) was denatured and size-fractionated on a 1% denaturing gel. Following electrophoresis, the RNA was transferred onto a membrane (Amersham) and immobilized by UV crosslinking. Probes were produced by random primer DNA labeling kit ver.2 (TAKARA) with templates from a 0.6-kb cDNA fragment corresponding to the 5′-region of coding sequence or 3′-UTR region. Hybridization was performed with ExpressHyb solution (BD bioscience) at 68 °C for 1 h and the membrane was washed twice in 2× SSC/0.05% SDS for 10 min at room temperature (RT) and twice in 0.1× SSC/0.1% SDS for 10 min at 45 °C.
Production of anti-AHI1 monoclonal antibodies. To produce T7-tagged AHI1 fusion protein as an immunogen, a 1242-base pair DNA fragment of rat AHI1 cDNA (corresponding to amino acid residues 1–414) was ligated into pET23a(+) expression vector. Recombinant protein expression was confirmed by Western blot analysis with a polyclonal anti-T7 antibody (Koma biotech). The induced recombinant protein was purified by eluting from the preparative scale SDS–polyacrylamide gel with the Disc Preparative Electrophoresis Apparatus NA-1800 (Nihon Eido) according to the manufacturer’s instructions. The eluate (T7-AHI1 fraction) was dialyzed against PBS and used as an immunogen for the generation of antibodies.