The protein extract of tissue homogenate was resolved on a 15% polyacrylamide gel slab (6.5 cm X 10.5 cm X 0.075 cm) by the method of Laemmli. The proteins on the gel were stained with Coomassie brilliant blue dye or transferred to a nitrocellulose membrane. After transfer, protein blots were immunodetected by Western blot procedures, using the P12-induced antisera or the partially purified antibody as the primary antibody and goat anti-rabbit IgG conjugated with horseradish peroxidase as the secondary antibody.
The total RNA of fresh tissue was prepared by the acid guanidium thiocyanate/phenol/chloroform method. RNA samples were analyzed by separation on a 1.0% agarose/formaldehyde-containing gel, followed by capillary transfer to a nylon membrane, and were hybridized to 32P-labeled nucleic acid probes. 32P-Labeled random-primed probe was prepared with a Promega random-priming kit (Promega, Madison, WI) using a template of P12 cDNA (369 base pairs [bp]) or a cDNA segment of mouse p-actin gene inserted into a pGEM4Z vector. flovent inhaler
Preparation of Spermatozoa
Cells were extruded from the distal portion of the caudal epididymis of adult male mice in a modified Tyrode’s solution containing 125 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 0.36 mM NaH2PO4, 4.5 mM glucose, 0.09 mM pyruvate, 8.9 mM lactate, 0.5 mM taurine, 100 IU/ml penicillin, and 100 ^g/ml streptomycin at pH 7.4.