Figure 2.

Binding of the PP2A monoclonal antibody, 4b7, to the PP2A C subunit carboxy terminus is completely inhibited by methylation of leucine 309. A C subunit carboxy-terminal octopeptide carboxy methylated on leucine 309 was synthesized and high-pressure liquid chromatography purified. This peptide (0.25 μg) was demethylated by treatment with 0.1 N NaOH (+) for 5 min at 4°C and then neutralized while another 0.25 μg of peptide (−) was treated with an equivalent amount of preneutralized solution. The unmethylated and methylated aliquots of the peptide were then spotted onto nitrocellulose, and the membrane was probed with 4b7 monoclonal antibody. Even on long exposure, no reactivity with the methylated peptide could be detected, indicating that 4b7 is absolutely specific for unmethylated C subunit.