Ca2+ Imaging in Xenopus Oocytes
To monitor Ca2+ release in Xenopus oocytes, oocytes were loaded with Ca2+ green-1 dextran (70 kDa) at a final concentration of 20 ^M 4-10 h prior to experiments. Oocytes were imaged through a 4X lens using a Nikon EC800 microscope equipped with a PCM 2000 confocal scanner and the software Simple (Compix Inc. Imaging Systems, Cramberry, PA). Images were collected in the nonconfocal mode (no pinhole) with excitation at 488 nm. Emitted light was collected in a 30-nm bandpass centered around 520 nm. Before, during, and after injection of SF, images were collected every 20-50 sec.
Superose 12 and Hydroxyapatite Fast Protein Liquid Chromatography
The SF or pig testis extracts were loaded at 4°C onto a Superose 12 Hr 10/30 column and a 5-ml hydroxyapatite column using a fast protein liquid chromatography (FPLC) system. Proteins were eluted, collected, and tested for Ca2+ activity as previously described.
Proteins from pig testis extracts and pig SF were diluted in sample buffer, separated by 8% SDS-PAGE, and transferred to nitrocellulose membranes (Micron Separation, Westboro, MA) essentially as described for boar SF. The membranes were blocked with 6% milk in 0.1% Tween 20, incubated overnight with dilutions of primary antibodies at 4°C, then washed and incubated for 1 h at 4°C with a secondary horseradish peroxidase-coupled antibody. After several washes, membranes were developed using the a detection system according to the manufacturer’s instructions (ECL; Amershan, Arlington Heights, IL). The primary antibodies used to probe the blots were a mixed monoclonal antibody against bovine PLC71 (1:1000; Upstate Biotechnology Inc., Lake Placid, NY), a polyclonal antibody raised against a peptide corresponding to the 20 amino acids at the c-terminal end of human PLC72 (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA), and a polyclonal antibody raised against the C-terminal 157 amino acids of PLC84 (1:500), which recognizes all three splicing variants of this enzyme.