The tyrosine kinase Pyk2 plays a unique role in intracellular signal

The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca2+ influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. cell pellets were thawed, resuspended, and incubated for 30 min in ice-cold TBS (150 mM NaCl, 15 mM Tris-Cl, pH7.4) containing 100 g/ml lysozyme and a low concentration of protease inhibitors (200 M phenylmethylsulphonylfluoride (PMSF), 1 g/ml pepstatin A, 2 g/ml aprotinin, and 1 g/ml leupeptine). Sarkosyl (1.5%) and -mercaptoethanol (10 mM) CHIR-265 were then added for 15 min on ice. Once the incubation was complete, lysates were centrifuged for 45 min at 250,000g. The supernatants were removed and neutralized with 2% Triton X-100. Transient Transfection of PC6-3 Cells PC6-3 cells (supplied by Dr. S. Strack, University of Iowa) were seeded at 2.5106 cells per 100 mm dish in RPMI medium (RPMI 1640 supplemented with 5% horse serum, 5% fetal bovine serum, 5% calf serum, 0.5% penicillin/streptomycin, 1% glutamine, and 1mM sodium pyruvate). Cells were transfected with Lipofectamine 2000 when 80C90% confluent. Briefly, 30 g of DNA was added to serum-free Opti-MEM. An 8% Lipofectamine 2000 solution was made simultaneously in serum-free Opti-MEM. After 5 min at RT, the DNA mix was added to the Lipofectamine mix followed by a 20 min incubation at RT. The medium on the cells was then replaced with Opti-MEM followed by addition of the DNA/Lipofectamine solution. The dishes were gently mixed and incubated for 6 h. The medium was then replaced with RPMI containing serum. The cells were harvested 48 h post-transfection using a cell scraper and Triton X-100 homogenization buffer (1% Triton X100, 150 mM NaCl, 10 mM Tris-Cl, 20 mM EDTA, 10 mM EGTA, pH 7.4) containing protease inhibitors (here: 200 M PMSF, 1 g/ml pepstatin A, 20 g/ml aprotinin, 10 g/ml leupeptine, 8 g/ml calpain inhibitor I/II) and phosphatase CHIR-265 inhibitors (1 mM pervanadate, 25 M NaF, 25 mM NaPPi). The cells were then homogenized with a dounce homogenizer followed by centrifugation at 250,000g for 15 min. CHIR-265 Supernatant was removed and the total protein was quantified with a BCA assay. An equal amount of protein (25 g) was extracted with SDS sample buffer and loaded for SDS-PAGE and subsequent immunoblotting with phosphospecific pY402 Pyk2 antibody. The immunoblots were then stripped and reprobed for total Pyk2 using the monoclonal anti-Pyk2 antibody. Primary hippocampal culture production and maintenance Primary hippocampal cultures were prepared as described previously (Lim et al., 2003; Chen et al., 2008). Briefly, hippocampi from E18 embryonic Harlan Sprague-Dawley rats were removed and incubated in Hanks balanced salt solution (HBSS; Invitrogen) with trypsin (0.03%) for 15 min at 37C. The cells were then washed three times with HBSS followed by trituration to dissociate cells. Dissociated cells were counted and plated for immunofluorescence on glass coverslips (60,000 cells per 35 mm dish) for microscopic analysis or in 100 mm culture dishes (800,000 cells per 100 mm dish) for biochemical analysis. The cells were incubated in Neurobasal medium (Gibco) containing custom-made NS21 supplement(Chen et al., 2008), 0.6 mM glutamine, and 5% fetal bovine serum (Brewer et al., 1993). After 3C4 h, the incubation medium was replaced with serum-free medium, and cells were maintained at 37C in humidified air composed of 95% air and 5% CO2. One third of the medium was exchanged weekly. Transient Transfection of Primary Hippocampal Cultures Primary hippocampal cultures (15 DIV) were transfected using an adapted calcium phosphate protocol. The medium was replaced with freshly prepared Neurobasal medium containing NS21 30 min prior to transfection. The removed conditioned medium was then retained for use later in the procedure. DNA (5 g) was added to CaCl2 (200 mM). An equal volume of 2X BBS (final concentrations-140 mM NaCl, 0.75 mM Na2HPO4, 25 mM BES, pH 7.1) was added dropwise followed by immediate vortexing. The DNA/BBS mixture was then added dropwise to the neurons followed by gentle mixing. After a 3.5 h incubation, the medium was removed and the cultures were washed once with HBSS (135 mM NaCl, 4 mM KCl, 1 mM Na2HPO4, 2 mM CaCl2, 1 mM MgCl2, CHIR-265 10 mM glucose, and 20 mM HEPES, CHIR-265 pH 7.35). Immediately hSNFS following the wash, the conditioned medium was added. The cultures were allowed to express protein for 72 h at 37C in humidified air composed of 95% air and 5% CO2 followed by use for immunofluorescence. Infection of Primary Hippocampal Cultures Primary hippocampal cultures (15 DIV) were infected using FIV carrying GFP-tagged PSD-95.