Identification of isoform-specific components of PP2A–B56 complexes. (A) HeLa cells treated with control and B56 regulatory subunit siRNA for 48–96 h were stained for DNA. Arrows indicate mitotically arrested, micronucleated, and binucleated cells. (B and C) Bar graphs show the frequency of binucleate (B) and micronucleated cells (C; means ± SEM, n = 2 measuring >200 cells per experiment). (D) Blots to confirm depletion of endogenous B56 regulatory subunits. KIF4A was used as a loading control and marker for mitosis. (E) Thymidine-synchronized HeLa cells were transfected with GFP-tagged PP2A–B56 regulatory subunits for 18 h, and then, the thymidine was washed out to allow the cells to enter mitosis. After 10 h, these cells were stained for DNA, mouse anti–Aurora B, and sheep anti-MKlp2. PP2A–B56 was visualized by GFP fluorescence. Arrows mark PP2A–B56 associated with the central spindle. (F) PP2A–B56 and –B55α complexes were isolated using GFP antibodies from metaphase (M) and anaphase (A) HeLa cells. Untransfected cells were used as a negative control (Mock). The immune precipitates (IP) were blotted, and the blots are representative of four independent experiments. siControl, control siRNA.