Fig 5. In vivo demethylation of sperm PP2A.
(A) Caudal sperm were incubated with 1 mM each of L-Homocysteine and Adenosine (L-Hcy + Ado), 1 mM L- Homocysteine (L-Hcy) or 1 mM adenosine (Ado) for 10 min. Sperm extracts were prepared by sonication and analyzed by western blot (2X106 sperm/lane) with anti-demethyl PP2A antibody and a duplicate blot probed with anti-PP2A antibody. Significant increase in levels of demethyl PP2A is observed on treatment with L-Homocysteine and adenosine (L-Hcy + Ado) compared to the untreated control sperm. (B) PP2A in extracts of L-Homocysteine and adenosine treated sperm was concentrated by microcystin pull down followed by western blot analysis of with anti-phosphotyrosine-PP2A (Y307) antibody shows increased levels of phosphorylated PP2A. (C) Caudal sperm incubated with 5 nM okadaic acid (OA), a concentration to specifically inhibit PP2A, also elevates demethyl PP2A and tyrosine phosphorylated PP2A as seen in (D) with 107 sperm/lane.