After thawing, lysates were boiled for 3 min and sonicated. at The Rockefeller University or college (New York, NY) (Czernik et al., 1991; Benfenati et al., 1992, 1996; Vaccaro et al., 1997; Menegon et al., 2000). Conjugation of the RU19 antibody with the fluorescent dye Cy3 and purification of the labeled antibodies were performed with the SJA6017 FluoroLink Cy3 kit and the Hitrap affinity column (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The following antibodies were purchased from your indicated sources: anti-CaMKII-specific (clone 6G9) mouse monoclonal antibody (Affinity Bioreagents, Golden, CA); anti-CaMKII-specific (clone CB-1) mouse monoclonal antibody (Zymed, San Francisco, CA); anti-tau-1 antibody (Boehringer Mannheim, Mannheim, Germany); peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies (Bio-Rad, Hercules, CA); FITC-conjugated goat anti-mouse, tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit, FITC-conjugated donkey anti-rabbit, 7-amino-4-methylcoumarin-3-acetic acid-conjugated donkey anti-mouse, and real rabbit IgG antibodies (Jackson ImmunoResearch, West Grove, PA). The enhanced chemiluminescence SJA6017 detection system was from Amersham Biosciences, and the BCA protein assay reagent was from Pierce (Rockford, IL). Ionomycin, norokadaone, okadaic acid (OA), and cyclosporin A were from Calbiochem (La Jolla, CA). All chemicals were of the highest grade available. Low-density main cultures of hippocampal neurons were prepared from embryonic day 18 rat embryos (Charles River, Calco, Italy), essentially as explained previously (Banker and Cowan, 1977; Verderio et al., 1999a). Hippocampi were dissociated by a 15 min incubation with 0.25% trypsin at 37C, and cells were plated at a density of 20,000C30,000/cm2 on poly-l-lysine (1 mg/ml)-treated glass coverslips in MEM (Invitrogen, San Giuliano Milanese, Italy), supplemented with 10% horse serum (Hyclone, Logan, UT), 2 mmglutamine (Biowhittaker, Ververs, Belgium), and 3.3 mm glucose. After allowing neurons to adhere to the Rabbit Polyclonal to GPR37 substrate for 3C4 hr, coverslips were transferred to dishes made up of a monolayer of cortical astrocytes (Booher and Sensenbrenner, 1972), without physical contact between neurons and glial cells. Cells were then managed in serum-free MEM supplemented with 1% N2 product (Invitrogen), 2 mmglutamine (Biowhittaker), 0.1% ovalbumin (Sigma, St. Louis, MO), 4 mm glucose, and 1 mm sodium pyruvate. Main cultures of cerebellar granule neurons were prepared from postnatal day 5 rats (Charles River) as explained previously (Gallo et al., 1987; Menegon et al., 1997). To favor neuronal survival and prevent glial cell proliferation, the medium was supplemented 3 d after plating with 10 mm KCl and 7.5 mcytosine-1–d-arabinofuranoside (Sigma). Cerebellar granule neurons at 12 d(DIV) or hippocampal pyramidal neurons at 14 DIV were rapidly washed with KrebsCRinger’s answer buffered with SJA6017 HEPES (KRH; in mm: 150 NaCl, 5 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES/Na, pH 7.4); supplemented with 2 mm EGTA; solubilized by scraping with solubilization buffer (1% SDS, 2 mm EDTA, and 10 mm HEPES/Na, pH 7.4); and immediately frozen in liquid nitrogen. After thawing, lysates were boiled for 3 min and sonicated. Equivalent amounts of protein were subjected to SDS-PAGE (Laemmli, 1970) and transferred to nitrocellulose as explained previously (Towbin et al., 1979). Filters were blocked for 1 hr at room heat in TBS (in mm: 150 NaCl and SJA6017 100 Tris-Na, pH 7.5), supplemented with 5% nonfat dry milk, incubated for 2 hr with either anti-/CaMKII (RU16; 1:10,000) or phosphospecific anti-CaMKII (RU48; 1:5000) antibodies in TBS/milk, washed five occasions for 5 min with TBS/0.1% Triton X-100, incubated for 1 hr with a peroxidase-conjugated goat anti-rabbit antibody (1:10,000) in TBS/milk, washed five occasions for 5 min with.