These data were confirmed by immunoprecipitation experiments in which sperm proteins obtained at 1- and 24-h capacitation were immunoprecipitated with cdlcp42 antibody and then blotted with PY20 antibody (Fig. 7A), stripped, and reprobed with alcp44 antibody (Fig. 7B). In these conditions, two tyrosine-phosphorylated bands of 42- and 44-kDa molecular mass corresponding to ERK-2 and ERK-1 (Fig. 7, A and B) were detected. This result is in agreement with those of Wang et al., who showed the presence of both ERK-1 and ERK-2 after immunoprecipitation with this antibody.
Incubation in capacitating conditions increased tyrosine phosphorylation in both proteins (Fig. 7A), as indicated the increase in the ratio between autoradiographic signals of PY20 and alcp44 decorated bands at 24-h versus 1-h capacitation (respectively, 1.7-fold increase for p44 and 4.6-fold increase for p42). For comparison, recombinant ERK-2 was run in the lane marked ERK-2rec (Fig. 7), showing a band at the expected 50-55-kDa molecular mass range. The presence of tyrosine-phosphorylated ERK-2 during capacitation was further confirmed by using an antibody directed against the tyrosine-phosphorylated form of ERK-2. buy levaquin online
FIG. 7. Immunoprecipitation of phosphorylated p42 and p44 ERKs in capacitating spermatozoa. The allcp42 antibody was used to immuno-precipitate (I.P.) p42 and p44 ERKs from spermatozoa at 1- and 24-h capacitation, and the blot was first probed with PY20 antiphosphotyrosine antibody (immunoblotting = I.B.) (A), then washed thoroughly and reprobed with alcp44 antibody (I.B.) (B). As control, 2 |j.g ERK-2 recombinant protein was run in the lane marked ERK-2rec, showing a band at the expected 50-55-kDa molecular mass. The protein band at about 55-60 kDa present in both blots is the heavy chain of the antibody used to immu-noprecipitate. Molecular weight markers (X 103) are indicated to the left of the blots. Fold increase in the ratio between autoradiographic signals of PY20 and alcp44 decorated bands at 24-h vs. 1-h capacitation was, respectively, 1.73 for the p44 and 4.6 for the p42. Representative of 2 similar experiments.