|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 9. Effects of Ht31 on LY294002-stimulated increase in sperm motility, RIIß localization and interaction with AKAP3 in spermatozoa. (A) Swim-up-selected spermatozoa were incubated for 1 hour in the presence of LY294002 (10 µM) with or without the active inhibitor Ht31 (10 µM) or the inactive molecule P-Ht31 (10 µM) and sperm forward and rapid motility was evaluated by CASA. Data represent means±s.e.m. of seven different experiments. *P<0.01 and **P<0.001 versus respective controls, #P<0.001 versus Ht31, Student's t-test for paired data. (B) Western blot analysis of insoluble protein extracts from swim-up-selected spermatozoa treated or not with 10 µM LY294002 in the presence or absence of 10 µM Ht31. Insoluble protein fractions separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane were revealed first with anti-RIIß antibody (upper panel) and, after stripping, with anti-ERK2 antibody to check for equal lane loading (lower panel). Human endothelial cells were used as positive control. Molecular mass markers are indicated to the right of the blot. Representative of three similar experiments. (C) Effects of Ht31 and LY294002 on RIIß-AKAP3 interaction as revealed by co-immunoprecipitation of RIIß with AKAP3. Swim-up-selected spermatozoa treated with Ht31 or LY294002 alone or in combination were extracted in immunoprecipitation buffer containing 0.1% SDS and immunoprecipitated with anti-AKAP3 antibody (see Materials and Methods). After 8% SDS-PAGE of the immunobeads, proteins transferred to nitrocellulose membranes were revealed with anti-FSP95 (upper panel) or anti-RIIß (lower panel) antibodies. Molecular mass markers are indicated on the left of the blot. Representative of two similar experiments.
|
