2.11 PP2A activity assay
The phosphatase activity of PP2A was evaluated using the PP2A activity
detection kit (R&D Systems, DYC3309-2) according to the manufacturer’s
instructions. Briefly, the cortexes or cells were lysed in lysis buffer
and centrifuged at 2000 g for 5 min. the protein concentrations
were measured using a BCA kit. Subsequently, the samples were added to
96-well plates coated with antibody specific for the PP2Ac. After
removing unbound stuff, a serine/threonine synthetic phosphopeptide
substrate, which is dephosphorylated by active PP2A to generate free
phosphate and unphosphorylated peptide, was added. The free phosphate
was detected by a dye-binding assay using malachite green and molybdic
acid after 30 min incubation. The phosphatase activity of PP2A was
evaluated by calculating the rate of phosphate release.