5and postnatal week 6 in Fig. the depolarization-dependent launch of NT-3 and BDNF from granule cells, leading to rules in cell differentiation and survival during cerebellar development. Fluorescent differential display (FDD) and full-length cDNA cloning were performed essentially as explained by Shiraishi et al. (1999). The FDD was performed using total RNAs from mouse (ICR, Nihon SLC, Hamamatsu, Japan) cerebella at eight different developmental age groups: embryonic day time 18 (E18), P0, P3, P7, P12, TSHR P15, P21, Z-YVAD-FMK and P56. The cDNA sequence of a1803/CAPS2a (GenBank accession quantity “type”:”entrez-nucleotide”,”attrs”:”text”:”AB098623″,”term_id”:”39840796″AB098623) Z-YVAD-FMK was identified using a DNA sequencer (Prism 3700; Applied Biosystems, Foster City, CA). In situ The cDNA sequence corresponding to amino acids 18-89 of a1803/CAPS2 was used as the template to prepare the antisense riboprobe. Cryosections of mouse mind (14 m solid) were fixed in 4% paraformaldehyde for 5 min, washed twice in PBS, and treated with freshly prepared 10 g/ml proteinase K (Invitrogen, Carlsbad, CA) at space heat. After acetylation, sections were incubated in hybridization buffer comprising 0.2 g/ml digoxigenin-labeled riboprobes at 43C overnight inside a humid chamber. Hybridized sections were washed by successively immersing in 4 SSC (in mm: 150 NaCl and 15 sodium citrate, pH 7.0, space heat), 2 SSC containing 50% formamide (50C, 30 min), 2 SSC (37C, 10 min), 2 SSC containing 20 g/ml RNase A (37C, 30 min), 2 SSC (37C, 20 min), and 0.1 SSC (space temperature, 30 and 5 min). The hybridization signals were detected with the digoxigenin detection (Roche Diagnostics, Indianapolis, IN). Rabbit and guinea pig polyclonal anti-a1803/CAPS2 and anti-CAPS1 antibodies were raised against the glutathione For immunoelectron microscopy of paraformaldehyde-fixed sections, mice (postnatal day time 15 in Fig. 5and postnatal week 6 in Fig. 5Homogenates of P21 mice cerebella were centrifuged at 100,000 (Shiraishi et al., 1999), and the pellet portion was further fractionated by centrifugation in continuous sucrose gradient from 0.3 to 1 1.8 m. After centrifugation, the subfractions were taken from the top (portion 1) of the gradient to the bottom (portion 16). For immunoaffinity purification, 250 g of Z-YVAD-FMK superparamagnetic polystyrene beads coated covalently with sheep anti-rabbit or anti-mouse IgG (Dynabeads M-280; Dynal, Lake Success, NY) were incubated over night at 4C in phosphate buffer (PBS, pH 7.4, and 0.1% BSA) containing the primary antibody (4 g), rabbit anti-a1803/CAPS2 (amino acids 235-556) antibody, mouse anti-synaptophysin antibody, mouse anti-CAPS1 monoclonal antibody, and control rabbit IgG and mouse IgG (Jackson ImmunoResearch, Western Grove, PA). P15 mouse cerebella were dissected and homogenized in low-sucrose homogenization buffer (in mm: 5 HEPES, pH 7.4, 5 EDTA, and 30 sucrose and protease inhibitor combination). Homogenates were centrifuged at 800 for 10 min, and the crude membrane in the supernatant was incubated over night with 500 g of the magnetic beads for preabsorption. The resultant supernatant-containing vesicle fractions were incubated with the primary antibody-bound beads in incubation buffer (PBS, pH 7.4, 5% fetal bovine serum, and 2 mm EDTA) for 1 hr at 4C. Vesicle-bound beads were collected and washed four occasions with incubation buffer and twice with PBS comprising 2 mm EDTA for 15 min each. The collected vesicles were analyzed by ELISA or Western blotting. The mouse NT-3 and BDNF cDNA were subcloned into the pEF4/Myc-His plasmid vector comprising the EF-1 promoter (Invitrogen). The a1803/CAPS2 cDNA was subcloned into the pcDNA3 plasmid vector comprising the cytomegalovirus (CMV) promoter (Invitrogen) to produce the pcDNA3-CAPS2 [crazy type (wt)]. The pcDNA3-CAPS2 (C2 + PH) experienced an internal deletion of amino acids 347-593, which corresponds to the C2 and pleckstrin homology (PH) website. The cDNA encoding the C2 and PH website (amino acids 344-598) was subcloned into the pCMV-hemagglutinin (HA) plasmid vector (Clontech, Palo Z-YVAD-FMK Alto, CA) to produce pCMV-HA-CAPS2 (C2 + PH), which expresses the HA-tagged C2 and PH website of a1803/CAPS2. A replication-deficient adenovirus Z-YVAD-FMK (Ad-CAPS2) was generated from the cosmid-adenovirus terminal protein complex method (Miyake et al., 1996). Briefly, the full-length a1803/CAPS2 cDNA was put into the CAG promoter manifestation unit of pAxCAwt cosmid cassette (Takara Bio, Otsu, Shiga, Japan). Recombinant viruses were generated by homologous recombination between Twenty-four hours after transfection with the.
5and postnatal week 6 in Fig