Figure 2. The PP6 holoenzyme is required for normal nuclear morphology. (A) PP6 complexes were purified from a cell line stably expressing PPP6C tagged at the C terminus with a FLAG epitope. The cells were arrested in mitosis using 200 ng/ml nocodazole for 18 h before cell lysis. The complexes were analyzed by mass spectrometry, SDS-PAGE, and Western blotting. Sequence coverage is based on the total number of peptides matching the protein found using Mascot, whereas peptide number is the number of peptides reported to be unique to this protein by MaxQuant and is thus a more stringent measure of identification for closely related proteins. (B and C) HeLa cells were transfected with control, SAPS, and ankyrin repeat domain siRNA duplexes alone or in combination for 48 h. The cells were Western blotted to verify depletion of the target proteins (B) or fixed and then stained with DAPI (shown), tubulin, and CREST antibodies (C). The graph shows the percentages of normal and micronucleated cells for the various conditions (n = 3). Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean. IP, immunoprecipitation. Bar, 10 µm. 1318 JCB • VOLUME 191 • NUMBER 7 • 2010