Supplementary Materials Supplemental Data supp_285_34_26269__index. A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis. cells (Stratagene, La Jolla, CA) was induced overnight at 25 C with 0.5 mm SP600125 supplier isopropyl 1-thio-d-galactopyranoside. Constructs encoding SNAP and NSF in pQE9 were generously provided by Dr. S. Whiteheart (University of Kentucky, Lexington, KY). The light chain of tetanus toxin (pQE3) was generously provided by Dr. T. Binz (Medizinische Hochschule Hannover, Hannover, Germany). These proteins were expressed as described (7). Purification of His6-tagged recombinant proteins was accomplished under native conditions according to QIAexpressionist. A pGEX-2T plasmid encoding human Rab3A was provided by Dr. P. Stahl (Washington University, St. Louis, MO). A plasmid encoding the C2B (residues 361C511) domain of rat synaptotagmin VI fused to GST was kindly provided by Dr. T. Sudhof (University of Texas Southwestern Medical Center, Dallas, TX). The C2B-T418E,T419E (C2BTE) mutant was obtained as described (9). The cDNA-encoding NFAT regulatory domain (residues 4C385) was generously provided by Dr. J. M. Redondo (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain). Expression in BL21 was induced overnight at 22 C with 0.5 mm isopropyl 1-thio-d-galactopyranoside, and recombinant proteins were purified on glutathione-Sepharose following standard procedures. Rab3A was always used prenylated and loaded with GTPS (6). Acrosomal Exocytosis in Permeabilized Sperm Human semen samples were obtained from normal healthy donors. Highly motile sperm were recovered following a swim-up separation in human tubal fluid as formulated by Irvine Scientific (Santa Ana, CA) supplemented with 0.5% CD68 BSA for 1 h at 37 C in an atmosphere of 5% CO2, 95% SP600125 supplier air. Concentration was adjusted to 5C10 106 cells/ml, and incubation proceeded for at least 2 h. Permeabilization was accomplished as described using 1.9 units/ml (6). Sperm were resuspended in ice-cold sucrose buffer (250 mm sucrose, 0.5 mm EGTA, 20 mm Hepes-K, pH 7) containing 2 mm DTT and treated as described in the figure legends. Acrosomal status was examined by staining with FITC-coupled agglutinin (30 min at 20 C, 50 g/ml in PBS). At least 200 cells had been scored for every condition. Adverse (no excitement) and positive (10 m free of charge Ca2+) controls had been contained in all tests. For each test, acrosomal exocytosis index ideals had been determined by subtracting the amount of reacted spermatozoa in the adverse control (range, 10C30%) from all ideals and expressing the ensuing ideals as percentages from the acrosome response seen in the positive control (range, 25C40%). The common difference between negative and positive settings was 13% (tests where in fact the difference was significantly less than 10% had been discarded). SDS-PAGE and Traditional western Blots The sperm had been cleaned in PBS, and protein had been extracted in ice-cold 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10% glycerol, 1% Triton X-100, and a protease inhibitor blend (P2714; Sigma). After sonication for 15 s (3 x with 10-s intervals) and incubation for 30 min at 4 C, the sperm components had been clarified by centrifugation at 14,000 for 20 min and utilized or kept at instantly ?20 C. The proteins from rat mind and human being sperm had been separated on 10% Tris-Tricine SDS-PAGE and used in 0.22-m nitrocellulose membranes (Hybond; GE Health care). non-specific reactivity was clogged by incubation SP600125 supplier for 2 h at space temperatures with skim dairy (5% for mind and 0.5% for sperm) dissolved in washing buffer (PBS, 0.1% Tween 20). The blots had been incubated using the monoclonal anti-calcineurin antibody (1:5000 in obstructing solution) over night at 4 C. Horseradish peroxidase-conjugated goat anti-mouse IgG (Kierkegaard & Perry Laboratories Inc., Gaithersburg, MD) was utilized as supplementary antibody (0.25 g/ml, in washing buffer containing 5% skim milk, 1 h at room temperature). Extra first and second antibodies were removed by washing five times for 7 min in washing buffer. Detection was accomplished with a chemiluminescence system (Western Lightning; PerkinElmer Life Sciences) and subsequent exposure to Pierce CL-XPosure film (Tecnolab) for 1C10 min. Indirect Immunofluorescence Nonpermeabilized sperm (7 106 cells/ml) treated as described in the figure legends were spotted on poly-l-lysine-covered slides and fixed in 2% paraformaldehyde in PBS for 10 min at 20 SP600125 supplier C. After fixation, the sperm were incubated in 50 mm glycine-PBS for 10 min at 20 C and permeabilized in 0.1% Triton X-100, PBS for 10.