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Fig. 10. RII overlay assay (A,B) and western blot analysis (C,D) of protein extracts from swim-up-selected spermatozoa treated or not with LY294002. After treatment with 10 µM LY294002, sperm samples were lysed and proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. RII overlay assay: For RII overlay assay, membranes were first incubated with purified RIIa (A) or RIIß (B) PKA regulatory subunits followed by probing with anti-RII
(A) or anti-RIIß (B) antibodies. After stripping, the same membrane was subsequently subjected to western blot analysis using PY20 antibody (C) and, finally, anti-ERK2 antibody (D) to check for equal protein loading (IB: western blot analysis). Arrowheads in A-C indicate the AKAP3, which is tyrosine phosphorylated following LY294002 incubation and which binds PKA RII regulatory subunits. Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.
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