Whole-cell protein lysates of cell lines were incubated either with 12CA5 antibody cross-linked to bovine serum albumin (BSA)–coated protein A–Sepharose beads (GE Healthcare) to immunoprecipitate HA epitope–tagged PP2A C, with 4 μg of clone 1D6 to immunoprecipitate endogenous C subunits, or with 50 μl of clone 2G9 hybridoma supernatant to immunoprecipitate B for 1 hour and subsequently incubated with 20 μl of protein G–Sepharose beads (GE Healthcare) for 1 hour. The immune complexes were washed once with IP Lyse, three times with tris-buffered saline [25 mM tris, 135 mM NaCl, 2.6 mM KCl (pH 7.4) with HCl]. A tenth of the immunoprecipitate was used for the phosphatase assays (for more details, see phosphatase assays section below), and the rest of the immunoprecipitate was boiled for 5 min at 95°C in protein sample buffer for immunoblot analysis.

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