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. 2015 Aug 27;6:8048. doi: 10.1038/ncomms9048

Figure 4. Purification of PP2A regulatory subunit.

Figure 4

(a) Set up of in vitro phosphatase assay. The substrate (Flag-S–ULK1) is stable in the reaction over time and dephosphorylated only in the presence of an enzyme source (total or fractionated cell extract). (b) Purification scheme for the identification of the PP2A regulatory subunit from starved ULK1/2 DKO cell lysate. (c) Example of phosphatase assay reaction during PP2A regulatory subunit purification. Fractions from Phenyl Sepharose column were assessed for activity against ULK1. Active fractions (15–18) were combined as input for the next purification step. (d) Silver staining (top) and phosphatase activity assay (bottom) of fractions from the final purification step. Active fractions are boxed in red. Indicated bands were excised for mass spectrometric analysis. (e) Western blot showing distribution of PP2AC, B55α and ULK1 S637 phosphatase activity after a Q-column.