Protein extracts were mixed with Laemmli buffer, boiled for 5 min, and electrophoresed on 8% SDS-PAGE gels. The gels were blotted to nitrocellulose overnight at 4°C using an electroblotter run with water-cooling at 200 mA in a Tris-glycine buffer (25 mM Tris, 192 mM glycine, 20% v:v methanol, pH 8.3). The blots were dried and blocked in 5% dry milk for 30 min at 22°C. Primary antibodies were diluted in 5% dry milk and incubated with blots overnight. The blots were washed twice in 5% dry milk + 0.05% Tween 20 for 20 min each and then in 5% dry milk for 20 min. They were then probed with either anti-rabbit or anti-mouse secondary antibodies conjugated to biotin; this was followed by washing as described above and by incubation with streptavidin-horseradish peroxidase conjugate. Labeled proteins were visualized by chemilu-minescence. Chemiluminescent reagent was prepared by dissolving 20 mg 3′-aminophthalhydrazide and 5 mg 4-io-dophenol in 0.5 ml dimethyl sulfoxide and adding 17 ml distilled H2O, 2.5 ml NaCl, and 5 ml 0.1 M Tris, pH 8.5. Right before use, 62.5 ^l H2O2 was added. Western blots were incubated in the reagent for 1-2 min and exposed to x-ray film. birth control pills
For reprobing with an additional antibody, the blots were stripped with 2.2 M glycine and 0.5 M NaCl for three washes of 20 min each, then washed in PBS with 10% BSA for 20 min, and finally washed two times, 20 min each, in PBS alone. The blots were blocked with 5% dry milk in distilled H2O and reprobed with primary antibody as described above.