Fig 2. Microcystin pulldown of sperm PP2A.
(A) Caudal sperm extracts (2X108 sperm/ml) were incubated with microcystin- sepharose beads. The microcystin- bound proteins were eluted by boiling with Laemmli buffer and analyzed by Western blot with Anti-PP2A antibody (MC). 10μl of the input caudal sperm extract was loaded as a positive control (Input). Flow through (FT) obtained following the pull down, i.e. sperm extract left behind after incubation with microcystin sepharose beads, shows negligible levels of PP2A at the same control input loading volume. The same extracts were run as a duplicate blot and probed with Anti-PP1γ2 as a control for microcystin pull down. (B) Extracts from 5X107 sperm from each region of epididymis were subjected to microcystin pull down. Equal amounts of the microcystin bound proteins boiled in SDS- sample buffer were loaded in each lane and probed with anti-demethyl PP2A antibody. A duplicate blot probed with anti-PP2A antibody as control.