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Fig. 11. Effect of inhibition of tyrosine phosphorylation of proteins by erbstatin on functional binding of RIIß to AKAP3 in human spermatozoa. Sperm proteins from spermatozoa treated with LY294002 (LY, 10 µM) or erbstatin (E, 25 µg/ml) alone or together for 1 hour, were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. (A) RIIß overlay assay: the membrane was incubated with purified RIIß subunit followed by probing with anti-RIIß antibody revealed by chemiluminescence. After stripping, the same membrane was subsequently probed with anti-AKAP3 (B, IB: AKAP3) and anti-FSP95 (C, IB: FSP95) antibodies. Arrowheads in A-C indicate the AKAP3, which is tyrosine phosphorylated following LY294002 incubation and which binds RIIß regulatory subunits. Anti-ERK-2 antibody was used to check for equal protein loading (D, IB:ERK-2). Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.
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